Studies have indicated expression of KCNQ channels in airway smooth muscle (ASM), a tissue that is predominantly regulated by muscarinic receptor signaling. Therefore, we investigated the function of KCNQ channels in rodent ASM and their interplay with Gq/11-coupled M3 muscarinic receptors. Perforated-patch clamp of dissociated ASM cells detected a K+ current inhibited by the KCNQ antagonist, XE991, and augmented by the specific agonist, flupirtine. KCNQ channels begin to activate at voltages near resting potentials for ASM cells, and indeed XE991 depolarized resting membrane potentials. Muscarinic receptor activation inhibited KCNQ current weakly (~20%) at concentrations half-maximal for contractions. Thus, we were surprised to see that KCNQ had no affect on membrane voltage or muscle contractility following muscarinic activation. Further, M3 receptor-specific antagonist J104129 fumarate alone did not reveal KCNQ effects on muscarinic evoked depolarization or contractility. However, a role for KCNQ channels was revealed when BK-K+ channel activities are reduced. While KCNQ channels do control resting potentials, they appear to play a redundant role with BK calcium-activated K+ channels during ASM muscarinic signaling. In contrast to effect of antagonist, we observe that KCNQ agonist flupirtine caused a significant hyperpolarization and reduced contraction in vitro irrespective of muscarinic activation. Using non-invasive whole animal plethysmography, the clinically approved KCNQ agonist retigabine caused a transient reduction in indexes of airway resistance in both wild type and BK β1 knockout (KO) mice treated with the muscarinic agonist. These findings indicate that KCNQ channels can be recruited via agonists to oppose muscarinic evoked contractions and may be of therapeutic value as bronchodilators.KCNQ (K K+ channel agonists may be useful as bronchodilators for asthma since ASM hyperpolarization by K+ channel openers can partly relax ASM  K+ channels  K+ channels in control of contraction of various smooth muscle cell types  following cleavage by phospholipase C (PLC); in addition, stimulation of a number of other PLC-coupled receptors also modulate KCNQ channels via multiple intracellular mechanisms  acts in those cells, at least in part, by modulation of KCNQ channel activity, however, this hypothesis remains to be tested. Moreover, a number of KCNQ openers have been developed that were first targeted as anti-epileptics . We use KCNQ channel pharmacology to evaluate the functional consequences of KCNQ currents on voltage and contraction of rodent ASM. We also utilize BK channel β1 knockout (KO) mice to understand the complementary role of BK and KCNQ channels in ASM.Here, we address the role of KCNQ channels in control of membrane voltage and contractions of ASM. Whereas KCNQ channels have been shown to moderate muscarinic agonist-evoked contractions of ASM  and maintained as homozygous lines. Control animals used in these studies were 2–3 month old C57BL/6J mice strain from Jackson Labs, or 2–3 week old rats (Sprague Dawley) from Charles River Labs. For tracheal constriction studies we used previously published protocols , at 37°C. Resting tension was continuously readjusted to 10 mN for 1 h and then challenged with 67 mM K+ PSS twice or more until reproducible contraction responses were achieved. Subsequent experimental challenges with drugs were normalized to the constriction response to the 67 mM K+ PSS solution. In 67 mM K+ PSS, the K+ reversal potential is depolarized and therefore K+ currents are unlikely to play a role in controlling membrane potential and contraction tone. For experiments that involve two muscarinic challenges, we found that the second challenge does not show significant fatigue . On average, the second challenge shows less than 1% reduction in response from that of first challenge for WT and β1 KO trachea, respectively. Normal PSS used was (mM) 119 NaCl, 4.7 KCl, 2.0 CaCl2, 1.0 KH2PO4, 1.17 MgSO4, 18 NaHCO3, 0.026 EDTA, 11 glucose, and 12.5 sucrose. The pH of the solution was adjusted to 7.35 by a 95% O2 - 5% CO2 mixture. The 67 mM K+ PSS utilized reduced sodium (56.7 mM NaCl) to maintain proper osmolarity. “Low K+” PSS solution was used to measure contractions in relatively hyperpolarized conditions. Low K+ PSS consisted of 1.0 mM K+ derived from the KH2PO4 in the PSS solution, with no added KCl. Normal PSS has a total 5.7 mM K+ derived from 4.7 mM KCl plus 1.0 mM KH2PO4. All other ingredients were unchanged.The BK channel β2+-free HEPES-buffered Krebs solution . After addition of 2.5 U/ml papain (Worthington), 1 mg/ml BSA fraction V, and 1 mg/ml dithiothreitol, TSM tissue was agitated at 37°C on a shaking platform (250 moves/min) for 20 min. Tissue was washed once with the Ca2+-free Krebs solution and digested with 12.5 U/ml of type VII collagenase  for 10 min on a rocking platform at 37°C. Digested pieces of tissue were washed three times in Ca2+-free Krebs-BSA solution by centrifugation (750 g for 2 min). The tissue was then triturated up to 5 min to disburse single tracheal myocytes. TSM cells were stored on ice in Ca2+-free Krebs-BSA solution and used the same day. A small (50 μ l) aliquot of the solution containing isolated tracheal myocytes was placed in an open 1.0 ml perfusion chamber mounted on the stage of an inverted microscope. The TSM cells were allowed to adhere to the glass bottom of the chamber for 20 min and then were perfused (2 ml/min) with Krebs solution.Tracheas were isolated as described above. The dorsal muscle layer was cut away from the hyaline cartilage rings and minced into ~1-mm pieces in Cam) and ionic currents of TSM cells were measured using perforated-patch whole cell recordings. Pipettes were pulled from borosilicate glass capillaries  using a Flaming/Brown micropipette puller P-97  and had resistances of 2–4 MΩ when filled with internal solution and measured in standard bath solution. Membrane current was measured with pipette and membrane capacitance cancellation, sampled at 100 μ s, and filtered at 2.9 kHz using an EPC-10 amplifier, and PATCHMASTER software . In all experiments, the perforated-patch method of recording was used with amphotericin B (600 ng/ml) in the pipette  according to previous procedures Lomask, . Eight t Lomask, , was meat-test. In the cases when more than two experimental groups were compared, a One-Way ANOVA was applied to determine variability among groups. A post-hoc Tukey HSD test was used to compare individual groups. For comparison of two groups with multiple factors, we used a Two-Way ANOVA to distinguish if the groups were different. The effects were deemed significant when a P < 0.05 was obtained. The results are expressed as the means ± standard error of means where applicable.Igor 5 , KaleidaGraph 4.1.1 (Synergy Software), and Excel 2007 (Microsoft Corp.) were used for statistical analyses. Significance was determined with paired or unpaired t levels of target genes normalized to that of β-actin.Tracheal muscle was dissected and pooled from either five wild type or five β1 KO mice. Rat tracheas were analyzed from four individual animals and expression values reflect mean data. The muscle RNA was extracted using Ambion Purelink RNA kit. One microgram of RNA was used for random primer, reverse transcriptase reactions using Applied Biosystems Reverse Transcriptase kit. 1/10 of the cDNA synthesis was used for SYBR green-based PCR amplification of cDNA using the forward and reverse primers listed in Table http://neuromab.ucdavis.edu/catalog.cfm). Guinea pig anti-KCNQ3 (1:1000) antibodies have been successfully utilized for staining of brain sections . The whole lungs were then fixed overnight in 4% paraformaldehyde/PBS, and washed in 20% sucrose/PBS for a second day. Frozen sections were cut in a sliding microtome at 25 micron thickness and immunostained using the free-floating section technique. Primary antibodies used were Neuromab monoclonal antibodies against KCNQ1 , KCNQ2 , and KCNQ4  and a non-commercial antibody against KCNQ3  that has been used successfully in the past  show greatest expression of KCNQ1. However, KCNQ5 is somewhat greater than KCNQ4 expression . We observed relatively strong staining of KCNQ1 and KCNQ4 proteins in the smooth muscle layer lining the airway  voltage commands and quantified the KCNQ current as that present during the last 400 ms of each sweep . Consistent with previous work, flupirtine roughly doubled the KCNQ current at potentials positive to −40 mV , and with flupirtine, they were −36 ± 1.4 mV and 9.1 ± 0.3 mV (n = 26), respectively, consistent with the biophysical and pharmacological behavior of KCNQ channels. Since the qRT-PCR data indicated subtle differences in the expression profile of KCNQ1–5 subunits across species, we also investigated KCNQ currents in mouse ASM, and saw a similar effect of flupirtine and XE991 on the currents attributed to KCNQ channels , which was increased to 16.8 ± 2.7 pA (n = 26) by flupirtine, but reduced to 0.5 ± 0.2 pA (n = 23) by XE991. In mouse ASM cells, the values were 6.4 ± 2.0 pA (n = 7), 13.2 ± 3.2 (n = 5) and 1.2 ± 0.6 pA (n = 4), respectively. Similarly, flupirtine shifted the V1/2 from −26 ± 2 mV, slope factor 9 ± 0.5 (n = 7) to −36 ± 1.7 mV, slope factor 8.9 ± 0.6 (n = 7). Thus, both rat and mouse ASM cells display KCNQ current of similar current amplitudes and pharmacological sensitivities.KCNQ currents were assayed in acutely dissociated tracheal muscle cells using perforated, whole-cell patch clamp recordings. The bath solution included the BK channel blocker paxilline (1 μ M) to enrich for the KCNQ current fraction. To estimate KCNQ currents in these experiments, we used two approaches. The first exploits the much slower kinetics of KCNQ current activation, compared to other Kp Figure . Indeed,p Figure . This cop Figure . At potep Figure , consist Figures , and shi Figures  , and 54 ± 6% (n = 5), respectively  and XE991 depolarized them by 4.7 ± 1.1 mV, relative to control (n = 6). The significant effect of flupirtine and XE991 is consistent with the significant KCNQ channel conductance  whereas there was no significant depolarization induced by XE991 in the presence of CCh. This suggests that although KCNQ channels control resting voltage, they may not be necessary for membrane repolarization during muscarinic-evoked contractions.We next tested whether the KCNQ channels also affect membrane voltage under mAChR stimulation. Given that KCNQ channel currents are largely active in the presence of CCh at its EC2 and M3 mAChR stimulation is the predominant mechanism mediating contraction of airway  . These results are consistent with KCNQ channels being required for control of membrane voltage at rest, but having a redundant role with BK channels during muscarinic signaling. On the other hand, the KCNQ agonist, flupirtine, hyperpolarized the membrane voltage under all circumstances , 112 ± 4% , and 135 ± 7% (n = 12) in control, flupertine and XE991, respectively. For mouse trachea, the percent of high-K+ contraction was 131 ± 3% (n = 3), 108 ± 2% , and 133 ± 4% (n = 3) in control, flupertine and XE991, respectively. The significant effect of the KCNQ-channel opener for both rat and mouse trachea suggests that at potentials where both KCNQ K+ and L-type voltage-gated calcium channels (VGCCs) channels are activated, M-channel openers can moderate ASM contraction. As a further test, when we used a low (1 mM) K+ external solution to hyperpolarize trachea membrane voltage negative to the voltage threshold for activation of L-type Ca2+ channels, there was no effect of flupirtine on the CCh-induced contraction , and in flupirtine, it was 71 ± 4% (n = 3). This is consistent with a specific effect of flupirtine on membrane voltage via KCNQ channels. In contrast to the effect of flupirtine, XE991 had no effect on CCh-induced contractions  at 3 Hz and 20 Hz, respectively, and in the presence of XE991, it was 22.7 ± 2.9% and 42.5 ± 3.9% (n = 5), respectively.The lack of effect of XE991 on CCh-induced contraction may be explained by a robust BK channel current that is sufficient to limit depolarization. To test this hypothesis, we compared the effect of XE991 on contractility of WT mice to mice with a genetically ablated BK channel β1 subunit. β1 KO mice have increased tracheal constrictions due to reduced activation of BK channels  and BK channel β1 KO mice to understand how muscarinic Gq-coupled signaling or redundancies with BK channels, respectively affect the role of KCNQ channels in ASM. We performed these tests at 0.5 μ M CCh, a concentration that produces half-maximal contractions, and at which there was no effect of XE991 both in WT and KO trachea  in the presence or absence of XE991, respectively, whereas in KO trachea, they were 94 ± 10.8% and 149 ± 4.9% , respectively. These results suggest that when BK channels are fully active, KCNQ channels may be functionally redundant in limiting contractions from membrane depolarization. However, when BK channel function is perturbed, as when the β1 subunit is lacking, KCNQ channels are essential in limiting depolarizations, which likely increase airway contraction via influx of Ca2+ through L-type VGCCs. These data further indicate that the lack of significant effect of XE991 at higher CCh concentrations in the KO trachea  in breathing mechanics that is correlated to increases of airway resistance . This contrasts with sympathetic neurons that show 80–90% KCNQ current inhibition at 10 μ M muscarinic agonist  on voltage or contractions during muscarinic signaling. Rather, the data indicate that functional redundancy with BK channels reduces the contribution of KCNQ channels during ACh-evoked contractions. At low muscarinic agonist concentrations, the BK β1 KO was sufficient to reveal effects of XE991. At higher muscarinic agonist concentrations the combination of β1 KO and M3 receptor blockade revealed a significant effect of KCNQ channels on membrane voltage  membrane potentials. However, muscarinic activation eliminated the effect of KCNQ blockade on membrane potential. While KCNQ agonist flupirtine relaxed muscarinic-evoked contractions, KCNQ blockade did not enhance it. This suggests that, although KCNQ channels can be pharmacologically recruited to relax airway, they do not contribute to relaxation during muscarinic-evoked contractions. In part, one might expect that the mechanism would be due to inhibition by signaling downstream of muscarinic receptor activation and that the muscarinic-evoked depolarization of ASM could be due to muscarinic inhibition of KCNQ channels. However, voltage clamp recordings indicate muscarinic agonist only weakly inhibits KCNQ channels (~19% inhibition at 0.5 μ M CCh, 46% at 10 μ M CCh Figure , approxi Figures  and cont Figures . Thus, win vitro constriction study supports a supplementary role for KCNQ channels in control of airway contractility when BK K+ channel activation is perturbed. This is further supported by in vivo experiments showing that RTG reduces the PenH index. Although the whole animal plethysmography may also reflect changes in breathing independent of airway resistance (Glaab et al., The + channel in lower ASM, whereas human lung may be more reliant on KCNQ channels. Further studies should elucidate the relative contribution of these two channel types in lower airway of rodents.This study in rodents support past studies showing KCNQ channel expression in ASM of guinea pig and human tissues (Brueggemann et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Inflammation plays a critical role in plaque initiation, progression, and disruption. As such, inflammation represents an emerging target for the treatment of atherosclerosis.We investigated the contributing factors for plaque enhancement and examined the relationships between regional contrast enhancement and the inflammatory activity of atherosclerotic plaques in an experimental rabbit model using contrast-enhanced high-resolution 3D black-blood magnetic resonance imaging (MRI) in comparison with histopathology.Ten atherosclerotic rabbits and three normal control rabbits underwent high-resolution 3D contrast-enhanced black-blood MRI. MR images and the corresponding histopathological sections were divided into four quadrants. Plaque composition was analyzed for each quadrant according to histopathological  and imaging criteria (enhancement ratio (ER), ER=SIpost/SIpre).A total of 62 non-calcified plaques  were identified based on histopathology. Mean ER values were significantly higher in atherosclerotic vessel walls than in normal vessel walls . Mean ER values were significantly higher in macrophage-rich plaques compared to the macrophage-poor plaques . Using multiple regression analysis, macrophage area and microvessel density were independently associated with ER values that reflected plaque enhancement (p <0.001).Contrast-enhanced high-resolution 3D black-blood MRI may be an efficient method to predict plaque inflammation.


Varicocele is a common pathology of the testis frequently associated with infertility. For its management, a fine morphological study of the testis, both macroscopically and microscopically, and an accurate choice of surgical procedure are mandatory. The present review focuses its attention on the anatomic substrates of adolescent varicocele and its pathophysiologic modifications. The comprehensive assessment of all the reported alterations should be considered by the clinician before deciding the type of treatment and the timing. The study of history of medicine has always shown a tight relationship between anatomy and surgery. If anatomy, in the ancient times, has provided knowledge for operative surgery thanks to the scalpel, in the modern times, the advent of instrumental investigations, both macroscopically and microscopically, helps surgeons define a full diagnosis of different pathologies and choose a proper surgical procedure with reduced surgical time and hospital length of stay and good cosmetic results. The present review shows the synergy between anatomy and surgery in the management of adolescent varicocele.Varicocele is a common pathology of the testis characterized by a varicosity of the pampiniform venus plexus of the spermatic cord, with an incidence in older adolescent of 12,4–17,8%, with an average of 14,7% . There iConsidering the predominance of the left side varicocele and the unique anatomy of the left testicular vein (LTV), many theories were postulated to explain the aetiology of varicocele: incompetence of the venous valve system , hydrostThe pathogenesis of testicular damage remains also controversial and many mechanisms have been proposed, including induced testicular hypoxia by venous stasis and small vessel occlusion, leading to Leydig cell and germinal cell dysfunction , retrogrThe testes are a pair of oval, slightly flattened bodies of whitish colour, measuring about 4 cm in length, 2–5 cm from before backwards, and rather less in thickness. Each testis is placed within the cavity of the scrotum in such a manner that its long axis is directed upwards and slightly forwards and laterally. Usually the left gland is a little lower than the right one .The testis is supplied by the testicular artery, a branch of the abdominal aorta. It is a slender vessel which, after a long course from the abdomen to the scrotum, reaches the posterior border of the testis, where it breaks up into branches . It has The veins running along the septula and those of the tunica vasculosa converge on the posterior border of the testis where they form a dense plexus, called the pampiniform plexus (PP), which finally pours its blood through the TV, on the right side, into the inferior vena cava (IVC), on the left side, and into the left renal vein (LRV) . The oriThe lymph vessels of the testis pass upwards in the spermatic cord and end in the lymph nodes at the sides of the aorta and inferior vena cava below the renal veins .The nerves for the testis and epididymis accompany the artery and are derived through the aortic and renal plexuses from the tenth thoracic segment of the spinal medulla .μm, and their cytoplasm split up into two or more layers; they do not form continuous cell layers and are not completely covered by a basal lamina [μm and a surface area between 3970 and 4472 μm2, while the tubular diameter is between 158 and 200 μm2. In adolescent affected by varicocele, the lamina propria shows different degrees of thickening till a maximum value of 35 μm. This is the result of an increased deposition of extracellular components, starting from the innermost layer of collagen fibers extending to the outer extracellular layer [Another morphological remark is the microscopic investigation of the testicular parenchyma based on the numerous histological alterations observed in both intra- and extratubular compartments of varicocele testis. The human seminiferous tubules are formed by a very complex stratified epithelium containing spermatogenic cells in different stages of development and supporting cells, named the Sertoli cells (SC). In the angular interstices between various seminiferous tubules, there are Leydig cells (LC), which represent the endocrine component of the testis . Both SCl lamina , 39. Thel lamina , 41. In l lamina . Their il lamina . Finallyar layer . This coar layer . The incar layer . Aquaporins (AQP) are membrane channel proteins that facilitate rapid passive movement of water. They have been identified in different tissues and also in the testis. In particular in varicocele testes AQP-1 displays an overexpression at venular endothelial cell membranes together with a previously unreported positive expression at the cell membranes of the Sertoli cells, immature germ cells, and Leydig cells. The overexpression could suggest an involvement of such a protein in trying to balance the excessive endotubular and extratubular fluid excess due to varicocele . MoreoveIn the last years great importance has been given to the role played by NO in the regulation of male reproductive function and fertility, Leydig cell function, myofibroblast contraction, and hence tubular peristalsis; on the contrary its over production can be harmful for both testicular and sperm functions . In adolAnother important morphological study for varicocele diagnosis should be the semen analysis characterized by increased number of pathological sperm forms, decreased motility, and decreased sperm density and motility . UnfortuVaricocele should be evaluated in the standing position, in a warm room. The scrotum should be first examined to rule out evident discrepancy in the aspect with evident dilatations of the veins of the pampiniform plexus. Afterward the patient should perform a Valsalva manoeuvre. With the patient supine the palpation of the testes should be performed to check for testis consistency and volume discrepancy. The evaluation of varicosity, according to the most used classification proposed by Dubin and Amelar, should be graded as follows: I = small sized only palpable during Valsalva manoeuvre, II = medium sized palpable at rest, and grade III = large sized visible at rest .Secondary varicocele can be caused by serious conditions, like tumors, and should always be considered, especially if evident in the less common right side or if does not change, rapidly, its size from the upright to the supine position.In addition, varicocele can be classified according to the degree of reflux identified by colour Doppler ultrasound scan: grade I = reflux induced by Valsalva manoeuvre with pattern 1, only very little reflux at the beginning of the Valsalva, or pattern 2, reflux during the full length of the Valsalva; grade II = intermittent spontaneous venous reflux, and grade III = continuous spontaneous venous reflux . DopplerVenography can identify the enlargement of the pampiniform plexus with reflux of blood into its tributaries. In addition, it can identify collateral vessels and incompetent valves. However this method has widely been replaced with other tests that are less invasive, less time consuming and determine lower exposure to radiation .Another fundamental morphological consideration for varicocele management is the evaluation of the testicular volume; in fact significant testicular volume loss is observed in varicocele testes. Testicular volume during preadolescents is constant and at the onset of puberty the testis suddenly increases in size even prior to other pubertal changes. In adolescents with a varicocele the rapid growth of the testis between the ages of 11 and 16 is effected by the varicocele and results in a volume discrepancy between the right testis and left testis ; moreoveOn the basis of their previous research, Kass and Reitelman suggested treating varicocele if one of the following conditions is present: (1) semen analysis is abnormal; (2) left testicular volume is at least 3 mL less than that of the right one or a more than 10% reduction in volume; (3) the response of either luteinizing hormone or FSH to Gn-RH stimulation is supranormal; (4) there are palpable varicoceles bilaterally; or (5) there is a large symptomatic varicocele . NowadayAside from the debate related to what cutoff to be used, there is a debate regarding how long patients with asymmetry should be followed up before deciding on surgery . RecentlThese findings have led to the common usage of testicular size as a surrogate for potential fertility. It is generally accepted that a normal testis volume differential is less than 2 mL or 20% . CurrentDifferent techniques have been proposed to treat varicocele in the adolescent age. These techniques can be divided into two different categories: (a) percutaneous embolization; (b) surgical procedures, open and laparoscopic. Percutaneous embolization can be performed both through a retrograde route via the femoral vein or an anterograde route via the incannulation of one of the dilated veins of the pampiniform plexus utilizing a small incision on the scrotum. These techniques can be performed as day case procedure with local anesthesia. The embolization can be performed with either a sclerosant agent or solid embolic devices under fluoroscopy.The open surgical procedures are the most used techniques to repair varicocele in the adolescent age. There are two classical routes to ligate the spermatic veins: (i) abdominal incision, with en mass ligation of spermatic artery and vein ; this technique has been nowadays substitute by the Palomo laparoscopic ligation; (ii) inguinal incision, to ligate the spermatic vein(s) in the inguinal canal (Ivanissevich technique). More recently the so-called subinguinal varicocelectomy has gained popularity. With this technique the spermatic veins are ligated, usually using magnification device, and the spermatic artery and lymphatics are spared. The inguinal approaches can be performed either under local or general anesthesia. With the development of laparoscopic techniques also in the pediatric population in the last decade the laparoscopic ligation of the spermatic vein and artery has been proposed with success. This technique should be performed under general anesthesia.The overall results of all the techniques described in adult have been recently reported in a meta-analysis. The higher recurrence rate is for the Palomo technique (14.9%), followed by the radiologic embolization (12.7%) and laparoscopic varicocelectomy (4.3%). The lowest recurrence rate is for the microsurgical inguinal technique (1.05%) followed by the inguinal Ivanissevich technique (2.63%). Hydrocele is usually a common complication particularly after the Palomo technique (8.24%), the Ivanissevich (7.3%), and the laparoscopic varicocelectomy (2.84%), but it is extremely rare after microsurgical varicocelectomy (0.44%). The overall spontaneous pregnancy rate is also significantly better after microsurgical varicocelectomy compared to other techniques .A recent meta-analysis and literature review performed on eleven studies published between 2000 and 2009, with a population of 1443 children and adolescents treated, have compared the two most popular approaches in the pediatric age group reported in the literature: the laparoscopic and the open techniques . Varicoc Other interesting data derived from the above meta-analysis is that the injection of blue dye before laparoscopic ligation significantly reduces the incidence of hydrocele formation and that ligation of both artery and veins during laparoscopic varicocelectomy significantly reduces the recurrence rate .There are very few reports on the effect of varicocele treatment on future fertility in the pediatric population. One recent paper has analyzed the long term (12–17 years) effects in terms of pregnancy rate with an overall success rate in 75% of treated patients with significant improvement in sperm count, abnormal forms, and viability compared to the preoperative examination .The present review has focused its attention on the anatomic substrates of adolescent varicocele and its pathophysiologic modifications. Considering that morphological and biomolecular alterations are already recorded in adolescent varicocele and could interfere with cellular migration, differentiation, and nutrition, it is important to consider the correction of varicocele, aside from the classical indications, also as an option to arrest the progressive damage that inevitably threatens for several years the adolescent testis. This comprehensive assessment should be considered when the clinician must decide the right operation at the best time for the correct patient.


Amyotrophic lateral sclerosis (ALS) is a fatal disorder of the motor neuron system with poor prognosis and marginal therapeutic options. Current clinical diagnostic criteria are based on electrophysiological examination and exclusion of other ALS-mimicking conditions. Neuroprotective treatments are, however, most promising in early disease stages. Identification of disease-specific CSF biomarkers and associated biochemical pathways is therefore most relevant to monitor disease progression, response to neuroprotective agents and to enable early inclusion of patients into clinical trials.CSF from 35 patients with ALS diagnosed according to the revised El Escorial criteria and 23 age-matched controls was processed using paramagnetic bead chromatography for protein isolation and subsequently analyzed by MALDI-TOF mass spectrometry. CSF protein profiles were integrated into a Random Forest model constructed from 153 mass peaks. After reducing this peak set to the top 25%, a classifier was built which enabled prediction of ALS with high accuracy, sensitivity and specificity. Further analysis of the identified peptides resulted in a panel of five highly sensitive ALS biomarkers. Upregulation of secreted phosphoprotein 1 in ALS-CSF samples was confirmed by univariate analysis of ELISA and mass spectrometry data. Further quantitative validation of the five biomarkers was achieved in an 80-plex Multiple Reaction Monitoring mass spectrometry assay.ALS classification based on the CSF biomarker panel proposed in this study could become a valuable predictive tool for early clinical risk stratification. Of the numerous CSF proteins identified, many have putative roles in ALS-related metabolic processes, particularly in chromogranin-mediated secretion signaling pathways. While a stand-alone clinical application of this classifier will only be possible after further validation and a multicenter trial, it could be readily used to complement current ALS diagnostics and might also provide new insights into the pathomechanisms of this disease in the future. Amyotrophic Lateral Sclerosis (ALS) is the most common fatal adult onset motor neuron disorder. Although some interacting pathomechanisms are already known, therapeutic options remain marginal With ALS diagnosis being based mainly on clinical and electrophysiological examination, serum and CSF analyses are only performed to exclude ALS-mimicking conditions. A major hindrance to the development of novel therapeutic strategies is that diagnosis usually occurs at a late disease stage, limiting the efficacy of neuroprotective approaches. It was shown that up to 10% of ALS patients die before achieving diagnostic certainty according to the revised El Escorial criteria These factors spur on an intensive search for diagnostic markers to enable earlier ALS detection, and help monitor disease progression and treatment response while furthering the understanding of the associated pathomechanisms. Despite notable contributions of CSF metabolomics profiling in ALS and considerable advances in mapping the normal human CSF proteome, the discovery of ALS-specific protein markers in CSF would allow the most direct clinical application In our study, primary CSF low molecular weight proteome profiles of 35 ALS patients and 23 age-matched controls were generated. To minimize pre-analytical variability, an immunodepletion of high-abundance CSF proteins was not performed. CSF was processed using chromatographically functionalized paramagnetic beads in combination with linear MALDI-TOF-MS (matrix-assisted laser desorption/ionization) to increase reproducibility, resolution and identification capabilities compared to SELDI-TOF approaches Primary CSF specimens from 35 ALS patients and 23 controls (main patient cohort) were included in this study. The ALS patients were diagnosed according to the revised El Escorial criteria 2/g) according to adapted manufacturer’s instructions. The protocol was adapted to 10 µl CSF and binding solution volumes, followed by the addition of 10 µl bead suspension. The dried bead eluates were mixed with alpha-cyano-4-hydroxycinnamic acid matrix solution and spotted onto a MALDI Anchor Chip target (Bruker). The analysis was performed with a linear Microflex MALDI-TOF mass spectrometer (Bruker) optimized for highly sensitive detection in the mass range up to 10 kilodaltons. The raw data used for statistical analysis and detailed patient information are available from http://www.mh-hannover.de/proteomix/plos/. Username and password will be provided upon request.In contrast to the proprietary chip-based SELDI-TOF MS analysis, we used superparamagnetic particles  to prepare CSF for MALDI-TOF-based proteomic profiling. CSF peptides and proteins were concentrated on the surface of weak cation exchange microparticles . Peptide fragmentation and detection were accomplished in the instrument’s LTQ ion trap. Proteins were identified using MS/MS Ion Search of the Mascot search engine  and the Entrez protein database .The identified proteins were further analyzed using enrichment analysis based on the DAVID bioinformatics online resource http://www.r-project.org). P-values of p< = 0.05 were considered significant, while p-values of p< = 0.1 were considered to show a trend towards statistical significance. The reported fold changes for the respective proteins of interest were calculated by dividing their mean concentrations in the ALS samples by the mean concentrations in the controls.The MALDI-TOF mass profiles were integrated into a multistep bioinformatics analysis. First, spectra were pre-processed using the PROcess R-package The expression of SPP1 was validated in the validation cohort using ELISA technology according to the manufacturer’s instructions  at a 75-fold dilution. CSF samples were randomly assayed in duplicate. Inter-assay variation was minimized by measuring three samples on different ELISA plates and subsequent normalization.Eight ALS, four of our controls and four additional control CSF samples , were tested against the CSFassay-human A.1.0 protein assay  http://www.mh-hannover.de/proteomix/plos/mascot. Username and password will be provided upon request. Performing a DAVID analysis on the full set of identified proteins, several functional groups were found to be significantly enriched in CSF ; sensitivity: 80.7% (286/350); specificity: 78.3% (197/250); AUC: 0.84. The results for the feature optimized model are presented as a receiver operating characteristic curve (ROC) in The proteins in the CSF matching the MALDI-TOF-MS mass peaks were alpha-1-antitrypsin (A1AT), cystatin C (CST 3), chromogranin A (CHGA), VGF nerve growth factor inducible protein (VGF) and the secreted phosphoprotein 1 (SPP1).To evaluate our results in a broader biochemical context, we performed a pathway-based analysis of the identified classifier peptides and proteins. The BisoGenet multi-tier Cytoscape application was used for visualization and analysis of biomolecular relationships involving A1AT1, CST3, CHGA, VGF and SPP1 A1AT  was found to be significantly (p< = 0.05) upregulated in ALS patients compared to the controls. Further, there was a trend (p< = 0.1) towards a lower expression of VGF, cystatin C (CST 3) and chromogranin A (CHGA). A higher expression of secreted phosphoprotein 1  was detectable in the ALS patients. VGF was already identified as potential biomarker in ALS, Since a CSF ELISA assay was only available for SPP1, we decided to further validate our findings in a quantitative mass spectrometry assay. . This assay allows the simultaneous measurement of the concentration of 80 proteins in human CSF samples. We used this procedure for 16 CSF samples. From the validation patient cohort, 8 ALS patient CSF samples were run together with four of our control samples and four normal human CSF controls (NextGen) in a single batch. All controls met precision specification goals of <30%. The results for our marker panel consisting of A1AT, CST 3, CHGA, and VGF were in line with the MALDI-TOF-MS data-derived ALS classifier. The fold changes in concentration for these proteins as well as for the 75 remaining analytes are listed in A definite diagnosis of ALS can, according to the diagnostic criteria in current use, be reliably made only in advanced disease stages. Laboratory and technical tests can help to exclude other ALS-mimicking syndromes but do not provide positive markers of the disease. It is therefore necessary to improve the sensitivity and specificity of the diagnostic tools. Moreover, the identification of disease-specific markers and their interaction in a common systems biological network could allow novel insights into disease-associated dysregulation of signalling cascades and more accurate monitoring of patients within therapeutic trials.Proteomics represents a combination of different methods for the study of peptides and proteins in tissues, biological fluids or cell cultures at a specific point in time ALS is a degenerative disorder of the central nervous system which primarily affects motor neurons in brain and spinal cord even though more widespread changes in brain regions other than the primary motor cortex have been detected by neuropathological and neuroimaging techniques. CSF may most probably reflect biochemical disease-related alterations as it is much closer to the central motor system than blood or urine. Analysis of the CSF proteome of ALS patients can identify pathophysiologically relevant pathways as well as non-specific changes associated with neurodegeneration in general. The protein component of CSF not only consists of brain-derived proteins but also of proteins abundant in plasma Cystatin C is widely expressed in neuronal and non-neuronal cells and physiologically acts as an inhibitor of extracellular cystein proteases. Its concentration in the CSF is about 5-fold higher than in serum, mainly resulting from secretion by the choroid plexus . VGF expression increases with neuronal activity, decreases with aging and is regulated by neurotrophic factors such as brain-derived neurotrophic factor (BDNF) . The decrease in VGF could therefore be the result of either neuronal loss/dysfunction and/or reduced growth factor secretion. In a transgenic ALS mouse model, VGF expression was reduced already in presymptomatic stages and decreased further with disease progression. In vitro, adenoviral VGF overexpression was protective against excitotoxic injury of primary spinal cord neurons VGF and CHGA belong to the chromogranin/secretogranin family of regulated neurosecretory proteins which can be detected in secretory large dense-core vesicles (LDCV) in neurons and neuroendocrine cells Alpha-1-antitrypsin is part of the serine protease inhibitor (serpin) family. It has been detected in amyloid plaques of Alzheimer’s disease patients, most probably as a result of increased astrocytic secretion SPP1 is an extracellular matrix protein first identified in osteoblasts but also expressed in neurons and glial cells. It is upregulated in acute and chronic inflammation, has been shown to possess neuroprotective capacities and was identified as a biomarker in the plasma of patients with relapsing-remitting multiple sclerosis Our study is therefore in line with previous findings in the CSF of not only ALS but also other degenerative diseases of the CNS. Therefore, it remains to be shown whether these members of our biomarker panel are specific for ALS or rather unspecific for different diseases associated with neuronal degeneration. As assessed by univariate analysis, two of the identified biomarker proteins (A1AT and SPP1) individually showed a significant change of expression in ALS patients.Although suspected to be non-specifically affected by different neurological diseases - diminishing their potential value as singular biomarkers for ALS - A1AT, CHGA, VGF and cystatin C together do, however, appear to be highly specific for ALS detection as a part of the condensed marker panel described in this study.Using a new mass spectrometry-based technique (MRM) for the quantitation of peptides and proteins in the CSF of our ALS patients, we were able to identify other proteins like cathepsin D (CTSD), thrombin (F2), plasminogen (PLG) and secretogranin III (SCG3) as key players in the biomarker network . These pCTSD is also a member of the cystatin family A further downregulated protein was SCG3 Further experiments with a strong aspect of quantitative analysis will be necessary to obtain a better understanding of the complex interactions involved in the onset of ALS. Moreover, to become useful as a diagnostic tool, CSF samples of patients in earlier disease stages and of patients suffering from ALS-mimicking conditions will have to be analyzed.In summary, this study shows that CSF proteome profiling using bead-based protein fractionation and MALDI-TOF mass spectrometry in combination with specialized data mining tools is able to identify biomarker patterns which have similarly been described in previous studies analyzing ALS CSF. While further studies are required to better understand the disease specificity of these markers, they ultimately might become valuable for improved diagnosis of ALS, monitoring of patients in clinical trials and increased understanding of underlying pathomechanisms. We built a validated protein network that will enable us to gain a deeper understanding of the complex nature of this disease. To identify enriched functionally-related protein groups, cluster these groups in terms of subcellular localization and map enriched protein groups to KEGG pathways, a DAVID analysis was performed for the 59 peptides and proteins that make up our pathway analysis-derived ALS proteome network. The results of this analysis are shown in Figure S1Venn diagram of identified CSF proteins. The Venn diagram shows that 44 of the 186 CSF proteins identified in this study were identified in ALS and control CSF alike while 62 were only identified in ALS and 80 in control samples.(TIFF)Click here for additional data file.Figure S2Boxplots. The four boxplots visualize the expression of A: CHGA, B: CST3, C: VGF 3689 Da peptide and D: VGF 3954 Da peptide in the main patient cohort.(TIFF)Click here for additional data file.Table S1CSF proteins identified by high-resolution LC-ESI-MS/MS.(DOC)Click here for additional data file.Table S2Comparison between MRM, MS/MS and BisoGenet pathway.(PDF)Click here for additional data file.Table S3DAVID analysis of the 59 proteins from the BisoGenet pathway.(DOC)Click here for additional data file.


Macrophage-CSF (M-CSF) is critical for osteoclast (OC) differentiation and is reported to enhance mature OC survival and motility. However, its role in the regulation of bone resorption, the main function of OCs, has not been well characterised. To address this we analysed short-term cultures of fully differentiated OCs derived from human colony forming unit-granulocyte macrophages (CFU-GM). When cultured on dentine, OC survival was enhanced by M-CSF but more effectively by receptor activator of NFκB ligand (RANKL). Resorption was entirely dependent on the presence of RANKL. Co-treatment with M-CSF augmented RANKL-induced resorption in a concentration-dependent manner with a (200–300%) stimulation at 25 ng/mL, an effect observed within 4–6 h. M-CSF co-treatment also increased number of resorption pits and F-actin sealing zones, but not the number of OCs or pit size, indicating stimulation of the proportion of OCs activated. M-CSF facilitated RANKL-induced activation of c-fos and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, but not NFκB nor nuclear factor of activated T-cells, cytoplasmic-1 (NFATc1). The mitogen-activated protein kinase kinase (MEK) 1 inhibitor PD98059 partially blocked augmentation of resorption by M-CSF. Our results reveal a previously unidentified role of M-CSF as a potent stimulator of mature OC resorbing activity, possibly mediated via ERK upstream of c-fos. Osteoclasts were completely absent in these mutant animals op/Csf1opCsf1), or administration of a neutralizing anti-CSF-1 antibody, displayed this high bone mass phenotype due to total lack, or significant reduction in the number of OCs op/Csf1opCsf1 background, or cessation of neutralizing anti-CSF-1 antibody administration. In both cases there were equivalent levels of OCs to those seen in control animals, leading the authors to deduce that the rate of bone resorption in animals with sub-optimal levels of M-CSF was slower.The critical role of macrophage-colony stimulating factor (M-CSF) in osteoclast (OC) differentiation from hematopoietic precursors was demonstrated in mice with osteopetrosis due to disruption of the CSF-1 gene (In short-term (6 h) semi-pure cultures of isolated neonatal rat OCs, human M-CSF enhanced survival, motility and cytoplasmic spreading, but inhibited resorbing activity, in a concentration-dependent manner; maximum effect at 0.5 ng/mL en masse by treating CFU-GM-derived cells with M-CSF and RANKL for 14d in situ for transcription factor activation and signalling phosphorylation assays. We demonstrate that although RANKL is necessary for resorption, M-CSF 10–25 ng/mL potently augments RANKL-induced resorption, an effect that is not due to enhanced survival but, rather, due to increased activation of resorption in OCs. We also demonstrate that M-CSF potentiates RANKL-induced c-fos activation and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, and that the resorption-stimulating effect of M-CSF is blocked by the mitogen-activated protein kinase (MEK) inhibitor PD98059. This is the first study to definitively identify M-CSF as a potent enhancer of resorption activation of mature OCs, a paradigm-shift for its role in physiology and pathophysiology.To address this knowledge gap, we established a mature OC model based on that of Fuller and colleagues −4 M 2-mercaptoethanol, 2 mM L-glutamine, 10 ng/ml recombinant human GM-CSF, 10 ng/ml IL-3, and 50 ng/ml stem cell factor) was purchased from Stem-Cell Technologies. Human M-CSF and neutralising polyclonal antibody against M-CSF was purchased from Chemicon. Primary rabbit monoclonal antibodies raised against β-actin, c-fos and total or phosphorylated forms of IκBα and ERK1/2 and HRP-conjugated anti-rabbit polyclonal antibody were from Cell Signaling Technology . Soluble RANKL coupled to GST fusion protein (RANKL) was generously provided by Drs Matthew Gillespie and Julian Quinn . Transcription factor activation kits incorporating detection antibody for the p65 subunit of Nuclear Factor kappa B (NFκB) and c-fos component of AP-1 were purchased from Pierce Biotechnology . TransAM assay kits for Nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) were from Active Motif .Eagle's MEM, penicillin/streptomycin, paraformaldehyde, Sigma Cell Dissociation Solution (1X) non-enzymatic (Cat. No. C5914), Fast Garnett GBC, MEK1/2 inhibitor PD98059 and naphthol AS-BI-phosphate were purchased from Sigma. Non-essential amino acids (100X) and FBS were purchased from Bovogen . Ficoll-Paque was purchased from Pharmacia Biotech. MethoCult GF H4534  were suspended in 3.0 ml Methocult GF H4534 in 6-well plates and incubated at 37C in humidified atmosphere of 5% CO2-air for 10 days. Pooled colonies (CFU-GM-derived OC precursors) were harvested into PBS. Precursors (6.5×106 cells/175 cm2) were then seeded into 175 cm2 tissue culture flasks. The cells were cultured in 20 mL of MEM containing 10% FBS, nonessential amino acids, penicillin 50 U/ml, streptomycin 50 µg/ml, 2 mM L-glutamine, M-CSF (25 ng/ml), and RANKL (125 ng/ml) for 14–21 days. The cultures were refreshed weekly by replacing additives in one half volume of media. Mature OCs were used in situ for signalling and transcription factor assays or dissociated and re-settled on dentine slices for survival and resorption assays.Collection of human umbilical cord blood, isolation of a mononuclear cell fraction, expansion of CFU-GM-derived OC precursors and differentiation of mature human OCs have been previously described 2 flask) for 30 min at 37C when most were detached by agitation. Remaining adherent cells were removed with a cell scraper. Detached OCs were washed, pelleted by centrifugation , resuspended in MEM/10% FBS and settled onto round dentine slices (6 mm diameter) in 96-well plates (n = 500–1000 OCs/well) and cultured for 4–72 h in 200 µL of media. Cells were fixed in 1% formalin and reacted for TRAP as previously described Mature OC cultures were treated with dissociation buffer  for 18 h in MEM/0.2%FBS and then treated with RANKL (125 ng/mL) or vehicle for 30 min. Nuclear extracts  were assessed for activation of NFκB(p65), c-fos and NFATc1 using ELISA-based transcription factor assay kits. Briefly, an oligonucleotide containing the consensus binding sequence for the relevant transcription factor is immobilised to a 96-well plate. Transcription factor contained in nuclear extracts binds specifically to this oligonucleotide and is detected through use of an antibody directed against either p65, in the case of NFκB, c-fos or NFATc1. Addition of a secondary antibody conjugated to horseradish peroxidise provides sensitive chemiluminescent or colorimetric readout that is quantified by spectrophotometry.2 flasks were serum-starved for 18 h when media was replaced with MEM/10%FBS with vehicle, RANKL (125 ng/mL), M-CSF (25 ng/mL) or both. Cytoplasmic extracts  were collected at 0, 5 min and 10 min. For c-fos expression, mature OCs were treated with or without M-CSF for 18 h before cell lysis. Total protein was subjected to SDS-PAGE on 4–20% gradient gels . Protein gels were transferred to nylon membranes  overnight at 4C  and blocked in PBS containing 5%BSA; 0.2% Tween 20 for 1 h. Immunodetection used primary rabbit monoclonal antibodies that recognised either total or phosphorylated forms of IκBα, ERK1/2, c-fos, or β-actin . Secondary antibody was HRP-conjugated anti-rabbit polyclonal antibody  A chemiluminescent substrate followed by autoradiography was used for detection . MCID software was used for densitometric analysis.Mature OCs in 25 cmData are expressed as the mean ± SEM where applicable. Differences between groups where determined using one-way ANOVA followed by Fisher's multiple comparison test, two-way ANOVA – general linear model (GLM) followed by Tukey's post-hoc test or unpaired T-test. Statistical significance was set at P<0.05. Treatment groups with annotations containing the same letter are not significantly different. We assessed the survival and resorbing activity of mature human OCs by seeding freshly harvested cells onto dentine slices and culturing them for 72 h. Survival of OCs cultured in media/10% FBS alone was enhanced (62% increase) by the addition of M-CSF (25 ng/mL) but more effectively so (165% increase) by RANKL (125 ng/mL). Neither M-CSF nor RANKL had any substantial effect on OC plan area (size). In the absence of RANKL, resorption was negligible. Activation of RANKL-induced resorption per OC was markedly enhanced (300% increase) by the addition of M-CSF (25 ng/mL) . In six In our 72 h cultures, we could not exclude the possibility that the observed resorption-stimulating effect was due to ongoing OC differentiation in the presence of M-CSF. In CFU-GM osteoclastogenesis cultures we previously found a linear increase in OC plan area over three weeks due to progressive cell fusion In mature human OCs, NFATc1 is not activated by either RANKL or M-CSF. Acute treatment with RANKL of OCs that had been serum-starved for 18 h produced a 320% activation of NFκB at 30 min . Pre-treSince MEK/ERK pathways are known to be involved in both RANK and c-fms signalling M-CSF alone decreased phosphorylated IκBα by 42% at 10 min, whereas RANKL exposure stimulated phosphorylation by 278%. There was no additional effect in combination with M-CSF. Total IκBα was unchanged over the time-course .The results above indicated that ERK1/2 phosphorylation was enhanced by co-treatment with M-CSF and RANKL, suggesting that MEK/ERK signalling may be involved in M-CSF enhancement of RANKL-induced resorption activity. To test this possibility, we examined the effects of PD98059, a specific inhibitor of the ERK1/2 activator MEK-1, on this effect. Mature OCs cultured on dentine slices were pre-treated for 1 h with PD98059 (5 µM), or vehicle, prior to treatment with RANKL alone or together with M-CSF for 72 h. Pre-treatment with PD98059 had no effect on OC survival, size or RANKL-induced resorption . In cultUtilising a human, mature OC activation assay, we have demonstrated that M-CSF effectively activates OC resorption, in addition to its roles in survival and motility. It is established that RANKL rapidly activates resorption in isolated mouse OCs Functional RANK is expressed on mature OCs and RANKL stimulates their activation Yao et al. Miyazaki et al. in vitro, independent of OC formation, is supported by data from two murine in vivo studies. The first investigated the capacity of cell-surface M-CSF (csCSF-1) to restore in vivo M-CSF function in the CSF-1-deficient osteopetrotic (op/Csf1opCsf1) background Our novel finding that M-CSF regulates the rate of osteoclastic bone resorption in vitro a three- to four-fold increase in resorption In healthy individuals, serum M-CSF increases from approximately 12 ng/mL in the early twenties to 20 ng/mL in the eighties, corresponding to the concentration range where we observed Karsdal et al. We hypothesize that incomplete blockade of c-fms signalling, or downstream signalling pathways, may provide a potential means to attenuate bone loss. In this situation, OC differentiation will proceed but the proportion activated to resorb will be lower, resulting in reduced bone resorption yet maintenance of OC-derived anabolic signals to osteoblasts.


In vitro, treatment of macrophages with immune complexes, but not oxidized low density lipoprotein, C-reactive protein, or TNF-α, induced a gene expression profile similar to that of the vulnerable plaques. That ligation of FcγR recapitulates the pattern of gene expression in vulnerable plaques suggests that the FcγR → macrophage activation pathway may play a greater role in human plaque vulnerability than previously appreciated.Stroke is a leading cause of death in the United States. As ∼60% of strokes result from carotid plaque rupture, elucidating the mechanisms that underlie vulnerability is critical for therapeutic intervention. We tested the hypothesis that stable and vulnerable human plaques differentially express genes associated with matrix degradation. Examination established that femoral, and the distal region of carotid, plaques were histologically stable while the proximal carotid plaque regions were vulnerable. Quantitative RT-PCR was used to compare expression of 22 genes among these tissues. Distal carotid and femoral gene expression was not significantly different, permitting the distal carotid segments to be used as a paired control for their corresponding proximal regions. Analysis of the paired plaques revealed differences in 16 genes that impact plaque stability: matrix metalloproteinases , MMP modulators , activating Fc receptors  and FcγR signaling molecules (higher in vulnerable). Surprisingly, the relative expression of smooth muscle cell and macrophage markers in the three plaque types was not significantly different, suggesting that macrophage distribution and/or activation state correlates with (in)stability. Immunohistochemistry revealed that macrophages and smooth muscle cells localize to distinct and non-overlapping regions in all plaques. MMP protein localized to macrophage-rich regions.  The morbidity and mortality associated with stroke results from the rupture of vulnerable carotid plaques; plaque removal by carotid endarterectomy significantly decreases stroke riskA major factor contributing to plaque rupture is degradation of the extracellular matrix. The matrix is predominantly collagen and elastinVulnerable plaques express elevated levels of MMP-1, MMP-8, and MMP-9 that co-localize with activated macrophagesThis investigation conforms to the principles outlined in the Declaration of Helsinki. Specimens were procured and processed according to approved IRB protocols. The study is classified as exempt category 4; no informed consent was necessary. Femoral and carotid endarterectomy tissue was provided by The Vascular Group, PLLC based at Albany Medical Center. Femoral plaques (n = 14) were extracted by longitudinal arteriotomy, usually as an adjunct to distal bypass; carotids (n = 16) were removed using the eversion method from symptomatic patients (3/16 or 19%) and asymptomatic patients (13/16 or 81%) with >60% stenosis. Tissue was collected sequentially over a 6-month period with the only criteria for exclusion being absence of a morphologically distinct distal and proximal segment, making it an inherently randomized study.-(Ct gene – Ct actin) where Ct represents the threshold cycle for each transcript. The limits of detection were set at Ct ≥35. As the amount of RNA recovered varied with the specimen, the genes were prioritized . Thus, the number of samples varies among the genes (n = 8–16).Immediately upon removal, the tissue was placed in RNAlater®  and transported to the lab for processing. Carotid plaques were divided into proximal  and distal . From each carotid region, two contiguous blocks were taken, one for paraffin embedding and one preserved in OCT  for immunohistochemistry. RNA was extracted from the remainder of the tissue. Proximal and distal carotid and femoral tissue was weighed and RNA extracted with Tri-reagent . Extraction was facilitated by homogenization with a Brinkman Polytron®. cDNA was prepared from RNA using iScript . qPCR primers  were desParaffin-embedded tissue was sectioned (7 µm), stained with Tri-chrome, and classified using the American Heart Association stability criteriaTissue lysates were prepared by homogenization and sonication in RIPA buffer  followed by ultracentrifugation . 3 µg protein/well was run on 10% gelatin gels according to manufacturers' directions ; 20 µg of HT15 media was used as the positive control 6) and differentiated THP-1 cells (1×106) were stimulated (18 h) with 200 ng/ml human TNF-α , 50 µg protein/ml oxLDL , 40 µg/ml C-reactive protein . The concentrations used reflect serum concentrations in patients with atherosclerosis 4 ; oxidation was confirmed using the TBARS assay with malondialdehyde as the standard Human monocytes, purified from peripheral blood by counter-flow centrifugal elutriation, were purchased from The University of Nebraska Medical Center. Elutriation separates the monocytes from other blood cells based on their sedimentation in a centrifugal field. The process uses physiological media, thus maintaining cell viability and function. Monocytes were differentiated  for 7 days. THP-1s, a human macrophage-like cell line (ATCC #TIB-202), were differentiated with PMA  before use. Monocytes-derived macrophages  for detection of differences between stable to vulnerable that are as large or larger than 120% of within-group standard deviation.Full demographic details of the 30 patients in the study are summarized in To mitigate against stroke, carotid endarterectomy of asymptomatic plaques with ≥60% stenosis is recommendedphox, PKC-δ, iNOS), the inhibitory FcγR IIb, and the scavenger receptor for oxLDL (SR-A). A qRT-PCR comparison between the femoral plaques and distal carotid plaque regions revealed no significant differences in expression of 20/22 genes  embedded in an extensive extracellular matrix (blue) , we predicted that they would contain more macrophages. Surprisingly, there was no significant difference in the expression of CD68 (macrophages) and α-actin (smooth muscle cells) between proximal and distal regions of carotid plaques  phox; PKC-δ and gp91phox were used as indicators of elevated NADPH oxidase. With the exception of MHCII, all of the genes were higher in proximal vs distal regions  are pro-inflammatory, expressing elevated levels of Fc regions . This gephox, PKC-δ) and increased MMP activity   . These rdecreased, arguing against SR-A/oxLDL signaling as a major contributor to matrix degradation. This finding is consistent with our results demonstrating increased expression of SR-A mRNA in femoral plaques. Although SR-A is increased, its ligation with oxLDL may not induce MMP production and the plaques remain fibrous.Macrophages can be activated by many of the constituents of plaques, notably TNF-α, oxLDL, C-reactive protein (CRP), and immune complexes (IC). To assess the effects of macrophage activation on the expression of the genes associated with vulnerable carotid plaques, differentiated THP-1 cells and monocyte-derived macrophages were treated with TNF-α, oxLDL, CRP, or IC and the resulting changes in gene expression were quantified. Of the 16 genes differentially expressed in proximal plaques , TNF-α iOf the four compounds tested, IC induced a pattern of gene expression most closely resembling that of proximal carotid plaque tissue. Fifteen of the 16 genes were similarly regulated in IC-treated THP-1 cells, 12 of 16 in primary macrophages . Of partIf FcγR ligation contributes to an inflammatory environment, conditions that increase FcγR expression and/or signaling would be expected to be pro-atherosclerotic. Indeed, monocytes from patients with acute coronary syndrome have significantly higher FcγR expression phox suggest that the proximal carotid plaque environment is conducive to M1 (inflammatory) macrophage polarization. Laser capture microdissection will enable macrophages to be recovered from plaques. Expression profiling of such macrophages will permit a comprehensive comparison of genes differentially expressed in macrophages from vulnerable and stable plaques and their comparison to the profile generated by activation of macrophages through the FcγR. However, the results presented herein raise the intriguing possibility that FcγR ligation of plaque macrophages contributes to plaque instability, a model that, to date, has received little attention.Admittedly, as macrophages in the plaque milieu are exposed to multiple stimuli this is an assessment focusing on the FcγR → MMP axis. Indeed, recent studies suggest that IC in conjunction with toll-like receptor ligation, may generate a protective response review, . HoweverTable S1Sequence of qRT-PCR primers used in these studies.(DOC)Click here for additional data file.Table S2Antibodies used in these studies. The isotype, company, and concentration used are listed. Note that Anti-MMP8, Anti-TIMP2, and control IgG 2a were used at 25 mg/ml.(DOC)Click here for additional data file.


Oleic acid has been shown to lower high blood pressure and provide cardiovascular protection. Curiosity arises as to whether super olein (SO), red palm olein (RPO) and palm olein (PO), which have high oleic acid content, are able to prevent the development of hypertension.p<0.001), 178.9±2.7 mmHg (p<0.001) and 167.7±2.1 mmHg (p<0.001), respectively, compared with SHR controls (220.9±1.5 mmHg). Bradycardia was observed with SO and PO. In contrast, the SBP and heart rate of treated WKY rats were not different from those of WKY controls. The SO and PO significantly reduced the increased heart size and thoracic aortic media thickness observed in untreated SHR but RPO reduced only the latter. No such differences, however, were observed between the treated and untreated WKY rats. Oil Red O enface staining of thoracic-abdominal aorta did not show any lipid deposition in all treated rats. The SO and RPO significantly raised serum alkaline phosphatase levels in the SHR while body weight and renal biochemical indices were unaltered in both strains. Serum lipid profiles of treated SHR and WKY rats were unchanged, with the exception of a significant reduction in LDL-C level and total cholesterol/HDL ratio (atherogenic index) in SO and RPO treated SHR compared with untreated SHR.Four-week-old male spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats were fed 15% SO, RPO or PO supplemented diet for 15 weeks. After 15 weeks of treatment, the systolic blood pressure (SBP) of SHR treated with SO, RPO and PO were 158.4±5.0 mmHg (The SO, RPO and PO attenuate the rise in blood pressure in SHR, accompanied by bradycardia and heart size reduction with SO and PO, and aortic media thickness reduction with SO, RPO and PO. The SO and RPO are antiatherogenic in nature by improving blood lipid profiles in SHR. Palm oil is rich in antioxidants and contains a high proportion of palmitic acid as well as considerable amount of oleic and linoleic acids SO and PO are obtained from fractionation, refining, bleaching and deodorization of crude palm oil Such increment in unsaturated fatty acid content in palm oil is desirable as epidemiological studies have demonstrated that dietary consumption of mono- and polyunsaturated fatty acids can decrease blood pressure (BP) and prevent the development of hypertension It has been suggested that the mechanism for the hypotensive effect of oleic acid is attributable to the increased concentration of oleic acid in lipid membranes that regulates the localization, activity and expression of important signaling molecules in the adrenergic receptor pathway, and enhances the production of vasodilatory stimuli while restricting the vasoconstriction pathways We, therefore, postulated that SO, RPO and PO which have high oleic acid content may possess potential antihypertensive activity and cardiovascular protective action. Although SO, RPO and PO possess the same major fatty acids, there are slight differences in their fatty acid composition, principally in the unsaturated to saturated ratio due to their different processing methods. In this study, we wished to examine this hypothesis by evaluating the effects of dietary intake of SO, RPO and PO on the systolic blood pressure (SBP) and heart rate (HR) in the spontaneously hypertensive rats (SHR) and its normotensive Wistar-Kyoto (WKY) control strain of four weeks of age for a duration of 15 weeks. Heart weight and aortic media thickness were examined as they are the cardiovascular parameters that are directly associated with increased BP. Blood lipid profiles and biochemical indices of liver and renal function were performed to assess whether these parameters were affected after long-term dietary intake of these oils.ad libitum access to food and drinking water throughout the experiments.All experimental protocols involving animals were in agreement with and approved by the Animal Care and Use Committee, Laboratory Animal Science Centre, Faculty of Medicine, University of Malaya. Three-week-old male SHR and WKY rats were obtained from breeding pairs purchased from BioLASCO Taiwan Co. Ltd. Animals were housed individually in individually ventilated cage (IVC) fitted with individual HEPA filter and bed-o’cobs® Laboratory Animal Bedding under constant temperature of 23.0±1.0°C and subjected to a 12-hour dark/light cycle (lights on at 0700 h). All rats had ad libitum for 15 weeks from 4 to 19 weeks of age. After 15 weeks of experiment, the animals were humanely sacrificed after a 12-hour fast by inhalation of an anesthetic agent, diethyl ether, achieved by placing the animal in a sealed container saturated with the anesthetic vapor. Blood samples were collected by exsanguination from anesthetized rats and centrifuged at 4000 rpm for 10 min. Serum was separated and stored in aliquots at −80°C until further analysis.The SHR and WKY rats were both randomly divided into four groups (n = 6 per group) and each group was fed either a standard rat chow diet (controls), 15% SO supplemented diet, 15% RPO supplemented diet or 15% PO supplemented diet SO, RPO and PO were supplied by the Malaysian Palm Oil Board (MPOB) together with the analysis of their fatty acid and phytonutrient composition . The fatSBP was measured by non-invasive tail cuff method using the NIBP controller  after the animals were pre-warmed at 38–40°C for 10 min Total serum cholesterol level was assayed using the cholesterol oxidase/peroxidase method, triglycerides was assayed using the lipase/glycerol kinase/glycerol phosphate oxidase-p-aminophenazone method, high density lipoprotein cholesterol (HDL-C) was determined by elimination method and low density lipoprotein cholesterol (LDL-C) was calculated using the formula, cholesterol - [(triglycerides/2.18)+HDL]. The serum was used to determine the biochemical indices of liver and renal function.The enface staining of the aorta with Oil Red O was used for gross determination of lipid deposition. The thoracic-abdominal aorta was cut open longitudinally with luminal surface facing up, pinned carefully on the surface of wax (Paraplast®) with stainless steel pins without stretching the tissue and fixed with 10% neutral buffered formalin. The inner aortic surface was stained with 0.3% Oil Red O  solution for 20 min at room temperature, and rinsed with 60% isopropanol and distilled water A 2.5 mm transverse section of thoracic aorta below the aortic arch was harvested using a cutting instrument for general histopathological studies, routinely processed and embedded in paraffin. Paraffin sections (3 µm) were cut and fixed on glass slides. The tissue sections were stained with hematoxylin  for 10 min, washed in running tap water, counterstained with eosin  for 6 min and washed with running tap water. Stained sections were dehydrated in 95% ethanol (2×10 sec) and 100% ethanol . Slides were cleared in xylene (3×2 min) and were mounted with DPX mountant  and topped with coverslips. Prepared tissue sections were examined under a light microscope (Leica DM750).p<0.05.Data are expressed as mean±SEM. The statistical analysis was performed using GraphPad Prism® 5 (GraphPad software). One-way or two-way ANOVA followed by Bonferroni’s multiple comparison test (where indicated) was used for statistical comparisons. Differences were considered statistically significant at 1,15 = 42.38, p<0.001), RPO  and PO  compared with SHR controls, when the whole duration of treatment was assessed by two-way ANOVA. Meanwhile, the HR of SO treated SHR , but not the PO  or RPO treated SHR , was significantly reduced compared with SHR controls  compared with SHR controls (p<0.01) than that of RPO treated SHR, but was not significantly different from that of PO treated SHR after 15 weeks of treatment. The SBP between PO and RPO treated SHR were also not significantly different from each other (p<0.01) and PO treated groups (p<0.05)  .In contrast, the SO, RPO and PO did not produce any significant changes in the SBP and HR of WKY rats over the whole duration of treatment compared with WKY controls .All the treated and untreated SHR and WKY rats did not show any significant differences in their daily caloric intake expressed in kilocalorie  .The average daily food intake for SHR controls was 18.49±0.56 g and SHR treated with SO, RPO and PO were 16.36±0.45 g, 16.78±0.43 g and 17.46±0.29 g, respectively. The average daily food intake for WKY controls was 15.76±0.57 g and WKY treated with SO, RPO and PO were 13.91±0.65 g, 15.40±0.96 g and 16.05±0.82 g, respectively.The average daily sodium intake for SHR controls was 0.0333±0.0010 g and SHR treated with SO, RPO and PO were 0.0245±0.0007 g, 0.0252±0.0007 g and 0.0262±0.0004 g, respectively. The average daily sodium intake for WKY controls was 0.0284±0.0025 g and WKY treated with SO, RPO and PO were 0.0209±0.0009 g, 0.0231±0.0014 g and 0.0241±0.0012 g, respectively.The body weights gained in treated animals over the 15 weeks treatment period were not significantly different from that of untreated animals . At the p<0.05 in both cases) but not in the RPO treated SHR (4.44±0.12) compared with SHR controls (5.00±0.35) (Heart weights were reduced in SO (1.45±0.04 g), RPO (1.45±0.04 g) and PO (1.38±0.04 g) treated SHR compared with untreated SHR (1.60±0.11 g) but the reductions in heart weight were not significantly different from the SHR controls . However00±0.35) .In WKY rats, significant differences in heart weight were not observed between the SO (1.02±0.02 g), RPO (1.09±0.07 g) and PO (1.12±0.03 g) treated groups and the controls (1.12±0.02 g) . Heart tp<0.001) in the SHR and not in WKY rats (p<0.01), RPO  and PO   . On the  p<0.01) . Enface p<0.01 in both cases) but not in PO (0.71±0.03 mmol/L) treated SHR compared with untreated SHR (0.82±0.06 mmol/L). Accordingly, the total cholesterol/HDL ratio of SO (3.2±0.07), RPO (2.9±0.06) and PO (3.6±0.32) treated SHR were lower than those of untreated SHR (4.2±0.27), but were statistically significant for both the SO (p<0.05) and RPO (p<0.01) treated SHR and not for the PO treated group. On the other hand, serum lipid profile of SO, RPO and PO treated WKY did not differ significantly from the untreated WKY.The levels of serum total cholesterol, triglycerides, HDL-C and LDL-C as well as total cholesterol/HDL ratio are presented in p<0.01) and RPO (p<0.001) treated SHR and not in the PO treated SHR. However, the levels of AST and ALT in SO, RPO and PO treated SHR were not significantly different from those of untreated SHR. On the other hand, the levels of ALP , AST and ALT in SO, RPO and PO treated WKY were not significantly altered compared with WKY controls. The total bilirubin and GGT for all treated and untreated SHR and WKY rats were below detectable levels.The levels of total protein, albumin, globulin, albumin/globulin ratio (AG ratio), total bilirubin, alkaline phosphatase (ALP), gamma glutamyl transferase (GGT), aspartate transaminase (AST) and alanine transaminase (ALT) in the serum of treated and untreated SHR and WKY rats representing the biochemical indices of liver function are presented in The SO, RPO and PO fatty acid composition and phytonutrient content are shown in The most common form of human hypertension is the primary or essential hypertension and is best represented by the genetic animal models of hypertension such as the SHR Normally, palm oil has approximately equal fraction of saturated fatty acid to that of unsaturated fatty acid In our study, SHR and WKY rats were fed with diet containing 15% wt/wt SO, RPO or PO where 34.7% of the total energy content was provided by fat, and this was similar and comparable to most human diets which contain 30–40% of the total energy content provided by fat The amount of sodium present in commercial rodent diets may raise concern. High sodium diet raises BP though such rise in BP augments renal sodium excretion and regains normal sodium balance. However, sodium retention coupled with high sodium intake can trigger a rise in BP in both susceptible humans and animals The amount of sodium present in the standard and formulated rat diets used in this study may still pose some concerns as to whether this may affect the outcome of our results, since the SO, RPO and PO diets formulated based on 15% wt/wt oil with standard rat diet contain a lower sodium content. The findings of Aoki et al. In this study, we demonstrated that the rapid and spontaneous rise in the SBP that occurs in the untreated SHR with age was effectively mitigated by dietary supplementation with SO, RPO and PO . These p2A/D-adrenoreceptor/G protein/adenylyl cyclase-cAMP/PKA pathway 2+, diacylglycerol and Rho kinase Oleic acid has been shown to lower the BP Palm oil also possesses abundant health promoting phytonutrients especially vitamin E and carotenes Bradycardia was associated with the fall in BP towards the end of the treatment period in SO and PO treated SHR . It is iCardiac hypertrophy is one of the features of hypertensive end-organ damage observed in the SHR and human essential hypertensives Our data showed that after 15 weeks of experiment, the heart weight and heart to body weight ratio of the SHR controls were higher than the normotensive WKY controls . This isHypertrophy and tissue disorganization are not limited to the myocardium but also involve vascular smooth muscle cells In our study, SHR controls exhibited significant aortic media thickening with evidence of increased muscle to elastic lamellae connections . SO, RPODespite the fractionation process in palm oil processing which brought about an increase in monounsaturated oleic acid with concurrent reduction in palmitic acid We showed that SO, RPO and PO treated SHR and WKY rats produced similar trend in their serum lipid profiles. Our data argue against the notion that long-term consumption of palm oil elevates the total cholesterol and LDL-C levels since the treated animals did not show any changes in the total cholesterol, and the LDL-C level was actually reduced with SO and RPO in the SHR . These fWe found that the biochemical indices of liver function were not affected following treatments with SO, RPO and PO in both the SHR and WKY rats, with the exception that the ALP levels were increased in the SHR treated groups. There was a tendency for the ALP levels to increase in the WKY treated rats. The elevated serum ALP levels may indicate cholestatic disorders or obstructive liver disease such as intrahepatic and extrahepatic obstruction to bile flow as well as extrahepatic biliary atresia although its precise roles are unknown After 15 weeks of experiment, the biochemical indices of renal function were determined for urea, creatinine and electrolytes to assess the functional capacities of the kidneys. Our results showed that long-term treatments with SO, RPO and PO did not produce any significant variations in the renal biochemical indices in both the SHR and WKY rats . In spitIn conclusion, our findings suggest that these oils can provide effective dietary interventions aimed at improved control of blood pressure in the treatment of hypertension. These oils also reduce the risk of cardiovascular disease associated with hypertension by improving the serum lipoprotein profiles, and cardiac and vascular smooth muscle hypertrophy produced by hypertension.


Doxorubicin (DOX), an anthracycline antibiotic is one of the most effective anticancer drug used in the treatment of variety of cancers .Its use is limited by its cardiotoxicity. The present study was designed to assess the role of a natural product resveratrol (RSVL) on sensitization of mammary carcinoma (Ehrlich ascites carcinoma) to the action of DOX and at the same time its protective effect against DOX-induced cardiotoxicity in rats.Ehrlich ascites carcinoma bearing mice were used in this study. Percent survival of tumor bearing mice was used for determination of the Cytotoxic activity of DOX in presence and absence of RSVL. Uptake and cell cycle effect of DOX in tumor cells in the presence of RSVL was also determined. Histopatholgical examination of heart tissues after DOX and/or RSVL therapy was also investigated.DOX at a dose level of 15 mg/kg increased the mean survival time of tumor bearing mice to 21 days compared with 15 days for non tumor-bearing control mice. Administration of RSVL at a dose level of 10 mg/kg simultaneously with DOX increased the mean survival time to 30 days with 70% survival of the tumor-bearing animals. RSVL increased the intracellular level of DOX and there was a strong correlation between the high cellular level of DOX and its cytotoxic activity. Moreover, RSVL treatment showed 4.8 fold inhibition in proliferation index of cells treated with DOX. Histopathological analysis of rat heart tissue after a single dose of DOX (20 mg/kg) showed myocytolysis with congestion of blood vessels, cytoplasmic vacuolization and fragmentation. Concomitant treatment with RSVL, fragmentation of the muscle fiber revealed normal muscle fiber.This study suggests that RSVL could increase the cytotoxic activity of DOX and at the same time protect against its cardiotoxicity. Doxorubicin (DOX) was introduced in cancer therapy in the late 1960s. It has emerged as one of the most potent broad-spectrum antitumor anthracycline antibiotics. DOX can be administered as a single agent or in combination with other chemotherapeutic agents. It is widely used in the treatment of variety of cancers, including leukemias, lymphomas, soft-tissue sarcomas, and solid tumors. Its cytotoxic effects on malignant cells, however, are complicated by an increase in the risk of cardiotoxicity .Due to the increasing worldwide prevalence and health burden of doxorubicin induced cardiotoxicity, it has become increasingly important to find pharmacological remedies to protect against this serious side effect, in an attempt to minimize DOX effective chemotherapeutic dose and thereby its side effects, a variety of approaches have been investigated -5. One oDoxorubicin hydrochloride and RSVL were purchased from Sigma–Aldrich Co. . The stock solution of both drugs were dissolved in normal saline and preserved at -20°C. The solutions were diluted in normal saline immediately before each experiment to the desired final concentration.6 cells/mouse. This study was approved by the ethical committee of King Abdulaziz Hospital.Female Swiss albino mice  were obtained from King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia. The animals were acclimatized for one week. A commercial balanced diet and water, ad libitum were provided throughout the experiment. A line of Ehrlich ascites carcinoma cells (EAC) cells was supplied by Prof. Abdel-Moneim and maintained in our laboratory by weekly I.P. transplantation of 2.5 × 106 cells/mouse). Twenty four hours later, mice were equally divided into four groups. Group 1: mice were administered normal saline i.p. (0.2 ml/20 gm) and served as control. Group II was administered DOX 15 mg/kg i.p. Group III received a single dose of RSVL  and Group IV received RSVL simultaneously with DOX.The effect of RSVL on the antitumor activity of DOX was evaluated using the modified regimen of Donenko et al. . In brie. Ehrich ascites cells were inoculated as described above at 10×106 cells/mouse. Twenty-four hours later RSVL (15 mg/kg) was injected i.p. simultaneously with DOX (20 mg/kg). Tumor samples were obtained at fixed times after DOX treatment, washed, counted, and DOX fluorescence intensity was measured at excitation and emission wavelengths of λ ex = 496 nm and λ em = 592 nm, respectively to determine DOX concentration.DOX cellular accumulation assessment in Ehrlich ascites cells was performed using spectrofluorometer  according to the method of Kitagawa et al. . Ehrich 6 cells/mouse). Twenty-four hours later, DOX  was injected simultaneously with RSVL . T umor cells were obtained at fixed times after DOX treatment. Cell cycle analysis was performed using flow cytometer  according to the method of Smets et al. [Ehrlich ascites cells were inoculated i.p.  were used as a control and received normal saline (i.p.) and RSVL . Group III received a single dose of DOX at dose of 20 mg/kg. Groups IV received DOX simultaneously with RSVL. Twenty four hours after DOX treatment, animals were anesthetized and heart specimens were fixed in 10% formalin for light microscopic study of the heart tissues.Statistical analysis was performed using SPSS . One way analysis of variance (ANOVA) followed by least significant difference (LSD) for post hoc analysis, was used for multiple comparisons. Statistical significance was acceptable to a level of p < 0.05.Table Figure 0/G1 phase after 24 hours, while DOX treatment showed 17 fold increase in the number of cells in G0/G1 phase with less number of cells in S phase  with reduced number of cells in S phase compared to DOX or RSVL.Treatment with RSVL induced accumulation of cells in Ge Figure . SimultaThree and twenty –four hours after DOX treatment the proliferation index (S phase + G2/M phase) inhibited by about 6 and 78%, respectively, whereas concomitant treatment with RSVL reduced the proliferation index significantly by about 29 and 85%, respectively Figure .Light microscopic examination of heart section of albino rats after a single dose of RSVL  showed a general architecture almost similar to control Figures  and 6. H2 total dose [2/M phase) of cells treated with DOX to 29 and 85% compared with 6 and 78% in cells treated with DOX alone, 3 and 24 hours after DOX treatment, respectively. This agrees with the uptake study, where DOX uptake was at highest level 3 hours after DOX treatment. Al-Shabanh et al. [2/M phases were more sensitive to DOX than cells in G1 phase [1/S regulators, such as cyclin D1 and cyclin E which are responsible for S phase entry [Doxorubicin is an excellent antitumor drug for treating several types of solid tumor, leukemia and lynphomas. However, acute or chronic toxicity is the dose limiting factor whereas, acute cardio toxicity represented mainly by transient symptoms, such as arrhythmias, while chronic toxicity can develop into irreversible cardiomyopathy, which affects approximately 30-40% of the patients who receive 500 mg/mmtal dose . In the tal dose . Our stutal dose ,14. Supph et al.  reportedG1 phase , howeverG1 phase -19 or inse entry ,21. Althse entry  This couse entry .RSVL which is known to display antitumor activity , has beeThe authors declare that they have no competing interest.AMO, SEA, OMA, HMA, ZAD, MNA and OHO sharing in experimental work and writing the manuscript. MFA did the flow cytometric analysis and interpreted the results and WSR investigated the pathological changes in the hearts together with NMA. All authors read approved the final manuscript.