Cardiac troponin detected with sensitive assays can be chronically elevated, in the absence of unstable coronary syndromes. In patients with chronic coronary artery disease, clinically silent ischemic episodes may cause chronic troponin release. T1 mapping is a cardiovascular magnetic resonance technique useful in quantitative cardiac tissue characterization. We selected patients with anatomically and functionally normal hearts to investigate associations between chronic troponin release and myocardial tissue characteristics assessed by T1 mapping.We investigated the relationship between cardiac troponin I concentrations and cardiovascular magnetic resonance T1 mapping parameters in patients with stable coronary artery disease enrolled in MASS V study before elective revascularization. Participants had no previous myocardial infarction, negative late gadolinium enhancement, normal left ventricular function, chamber dimensions and wall thickness.R = 0.33, P = .012) and ECV , and were independently associated with nativeT1 (P = .049) and ventricular mass index (P = .041) in multivariable analysis.A total of 56 patients were analyzed in troponin tertiles: nativeT1 and extracellular volume (ECV) values (expressed as means ± standard deviations) increased across tertiles: nativeT1  and ECV . Cardiac troponin I concentrations correlated with native T1(In patients with chronic coronary artery disease and structurally normal hearts, troponin I concentrations correlated with T1 mapping parameters, suggesting that diffuse edema or fibrosis scattered in normal myocardium might be associated with chronic troponin release. Different mechanisms of chronic troponin release have been described in patients with structural heart disease  and normal ventricular structure (inflammation and demand ischemia). Specifically in patients with chronic coronary artery disease, clinically silent ischemic episodes, due to small vessel occlusion and plaque microembolization may cause chronic troponin release. If these ischemic episodes would cause edema or focal areas of fibrosis, a very sensitive imaging method like cardiovascular magnetic resonance (CMR) could detect these subtle tissue alterations, and find tissue clues of chronic troponin release.In stable patients, detectable troponin concentrations  that corresponds to gadolinium distribution, enabling the detection of interstitial edema and fibrosis, and infarction areas in patients with chronic coronary artery disease. Therefore, T1 mapping and ECV can detect subtle alterations in otherwise normal hearts, differentiating between cellular and interstitial alterations. by T1 mapping has been shown to be relevant in risk stratification in chronic CAD patients,Late gadolinium enhancement (LGE) by CMR is an accurate method to detect myocardial fibrosis. The prognostic relevance of T1 mapping has also been demonstrated, in a 4-years follow-up study after acute ST-segment elevation myocardial infarction: both infarcted and noninfarcted T1 were independent predictors of MACE  and significantly improved risk prediction beyond left ventricular ejection fraction, infarct size, and microvascular obstruction.Prognostic relevance (above risk scores and left ventricular ejection fraction) of T1 mapping characterization of noninfarcted myocardium (areas without LGE) has also been demonstrated in chronic CAD patients: native T1 was an independent predictor of all-cause mortality and the association of native T1 (noninfarcted) and LGE (extent), in a model that avoided multicollinearity, was an independent predictor of MACCE .,6 T1 mapping in infarcted and noninfarcted myocardium areas has also been demonstrated to have prognostic relevance.,18 In structurally normal hearts, the mechanism of chronic troponin release and its prognostic relevance may be associated with scattered myocardial tissue alterations that can be assessed by CMR T1 mapping and were not previously appreciated with conventional methods. This possible association has not yet been investigated and may help to understand the anatomical substrate for troponin elevation in chronic patients.In summary, in stable patients, chronic troponin elevation is common, and may have prognostic value.The patients included in this study, were selected from the MASS V trial which compared biomarkers and CMR before and after coronary revascularization procedures. Patients included had multivessel CAD, stable angina symptoms, and normal left ventricular ejection fraction with a formal indication for revascularization. therefore, we excluded all patients with detectable LGE by CMR in this analysis (n = 42). Patients with hypertrophic, infiltrative, Chagas disease or any other kind of cardiomyopathy were also excluded.In the present study, we analyzed troponin I concentrations in association with CMR T1 parameters before the proposed intervention. Previous myocardial infarction was not an exclusion criterion in the MASS V study. A scar from a previous myocardial infarction and detectable LGE can alter troponin concentrations and ECV,The study conformed to the guidelines set out in the Declaration of Helsinki, and all participants gave written informed consent. The Institutional Ethics Committee of the University of São Paulo approved the study.Blood samples were collected from each patient before the revascularization procedure was undertaken. Glucose, total cholesterol, HDL-cholesterol, LDL cholesterol, triglycerides, High Sensitivity C-reactive protein, and CKMB were determined with standard procedures.th of a reference population. According to the manufacturers, the detection limit is 6 ng/L, and the population reference value at the 99th percentile is 40 ng/L, the coefficient of variation is < 10% at the 99th percentile. Siemens Advia Centaur ultra-troponin I kit is designated as a contemporary or level 1 troponin assay, corresponding to <50% of normal individuals with detectable troponin concentrations.Cardiac troponin I was determined using the ADVIA Centaur TnI-Ultra kit  in the automated equipment of the same manufacturer. The test is an immunoassay that uses a direct chemiluminescence technology and constant amounts of 2 monoclonal antibodies. An increased troponin I concentration was defined as a value exceeding the limit of detection of the method and below the 99CMR examinations were performed using a clinical 1.5T scanner . Standard cine-imaging with steady state free precession pulse was used to acquire left ventricle (LV) short-axis images for quantification of ventricular volumes, mass and function. Late gadolinium enhancement was also performed using standard segmented IR prepared gradient-echo sequence 10 to 15 minutes after an intravenous bolus injection (0.1 mmol/kg) of Gadoterate meglumine Gd-DOTA . T1 mapping was performed using an ECG-triggered single-shot modified look-locker inversion recovery sequence with a 3 (3) 3 (3) 5 sampling pattern and the following parameters: slice thickness 10 mm, field of view 300 × 300 mm, ACQ matrix (read-out × phase-encodings) 152 × 150, flip angle 40, minimum TI 60 ms, inversion-time increment 150 ms. A mid modified look-locker inversion recovery short-axis image was acquired prior, and 15 minutes after the intravenous bolus of contrast.CMR images were analyzed using the CVI42 software  by experienced imaging experts  who were unaware of clinical or laboratory data. End systolic, end-diastolic LV volumes, LV mass and LV ejection fraction were calculated using standard methods. For T1, a region of interest was manually drawn at mid-ventricular level, sufficiently far from tissue interfaces, for both myocardial and LV cavity blood, as depicted in Figure Variables are expressed as means ± SD and frequency (percentage). Normality assumption was assessed graphically using QQ plot. Patients were split in tertiles of troponin I concentrations. Differences across tertiles were assessed using 1-way analysis of variance (ANOVA) for normally distributed variables, Kruskal-Wallis for variables with non-normal distribution and chi-square for proportions. as well as for the significant variables found in the univariate model. Since native T1 and ECV are correlated (multicollinearity), results for both variables were analyzed in 2 different multivariable models. All statistical analyses were performed using R version 3.5.3  and a P value < .05 was considered statistically significant.Correlations between troponin I concentrations and T1 mapping parameters were determined using Spearman correlation coefficient. A multivariable linear regression analysis was performed to investigate the association between troponin concentrations and native T1/ECV adjusted for age and sex,From a total of 202 patients who underwent CMR, T1 mapping was performed in 98 patients and 56  with negative LGE were selected for the current study. A total of 37 (66%) of the patients had detectable troponin concentrations (above 0.006 mg/dL).P = .003). LV volumes and function were similar across tertiles. LV mass increased across tertiles with a marginally significant P = .062. ECV and native T1 (expressed as means ± standard deviations) increased in a stepwise fashion across cardiac troponin I (cTnI) tertiles: ECV  and nativeT1 , as illustrated in Figure Baseline characteristics across troponin tertiles are shown in Table R = 0.30, P = .025) and native T1 . As depicted in Table P = .049), native T1 (P = .012), and LV mass index (P = .014). In multivariable linear regression analysis troponin concentrations were independently associated with LV mass index (P = .041) native T1  but not with ECV (model 2).Figure The main idea of this study and its results are shown in Figure In the present study, we evaluated 56 patients with stable, multivessel CAD, normal ventricular chamber dimensions, normal wall thickness, and negative LGE by CMR. The specific contribution of this study is to show that among patients with structurally normal hearts, differences detected by T1 mapping CMR were still found across troponin tertiles as reflected by higher ECV and native T1 and an independent association with LV mass index and native T1. Therefore, focal areas of edema or fibrosis in myocardial cells or interstitium might be associated with troponin elevation.,25 In this study, we selected stable patients without detectable LGE and normal cardiac chambers size and function, to investigate troponin release in the absence of myocardial scars. In this setting, this is the first study to show an association between chronic troponin release, ECV, and native T1 by CMR.Previous studies that investigated T1 mapping and troponin analyzed patients with myocardial scars. In patients initially free of cardiovascular disease who underwent serial cardiovascular magnetic resonance with late gadolinium enhancement, there was an association of high sensitivity troponin T levels with progressive fibrosis. It is possible that the current study findings with T1 mapping detect the very initial phase of this fibrosis process. The mechanism of diffuse myocardial damage might also explain the independent association of high sensitivity troponin I levels and incident heart failure in the general population. Diffuse myocardial injury detected by T1 mapping is associated with subtle ventricular dysfunction in diabetic patients and diastolic dysfunction in those with hypertrophic cardiomyopathy, associations that might also help to understand the worse clinical prognosis of stable CAD patients even with minor troponin I releases. As a future perspective, studies in chronic patients that associate high sensitivity troponin, T1 and T2 mapping may improve risk stratification and help to predict prognosis in the long-term.Consistent with our findings, other studies have also found an association between myocardial damage detected with different CMR techniques and high sensitivity troponin elevation. In patients with hypertrophic cardiomyopathy analyzed with CMR T2-weighted imaging there was correlation between myocardial injury and high sensitivity troponin T levels. but also defines a subset of patients at a higher risk of cardiovascular events: chronic troponin elevation in diabetic patients is associated with the presence of CAD, with an increased risk of cardiovascular death, myocardial infarction and cardiovascular death, and with all-cause death, myocardial infarction and stroke in patients with limb ischemia with and without CAD. In this higher risk group of patients, there is an increase in native T1 and ECV which can be caused by the summed damage of CAD, in epicardial coronary arteries and microvascular disease associated with diabetes mellitus. A similar association was shown in another study in which a subset of patients with preserved ventricular function increased ECV and high troponin and brain natriuretic peptide concentrations were at higher risk of incident heart failure hospitalization, and death from any cause.Diabetes mellitus prevalence was higher in troponin tertiles 2 and 3 compared to tertile 1. This finding is consistent with the importance of diabetes mellitus as a predictor of chronic troponin elevation in the general populationAlthough ECV had a consistent correlation with troponin increase, it was not associated with troponin concentrations in multivariable analysis. The probable reason for this lack of association is the relatively small number of patients. However, it is also possible that chronic troponin elevation is more strongly associated with changes at the cellular level in cardiomyocytes, as reflected by increasing native T1. The lack of a T2 mapping sequence is a limitation of this study to a better interpretation of ECV. this is not an unexpected finding. However, it is important to note that ventricular mass measurements were within the normal range, which indicates that troponin is a biomarker of subtle alterations in myocardial structure.There was also a consistent association of troponin elevation with left ventricular mass. As 55% of the patients were hypertensive and ventricular hypertrophy is associated with troponin elevation,Limitations to this study are the small number of patients evaluated, and the relatively small number of patients with T1 mapping in the MASS V study. T1 mapping was not a predetermined measurement in the MASS V study, which used cardiovascular magnetic resonance with late gadolinium enhancement to detect myocardial infarction. Previous myocardial infarction was neither an exclusion criterion if it did not compromise ventricular function. These factors may have led to a relatively small final number of patients with T1 mapping and LGE negative CMR. but this range may be broader, between 22% and 32%, or narrower, between 21.7% and 26.2%. These values may vary according to the equipment and specific technique used. The lack of a control group to determine normal ECV values with the method used is a limitation of this study. Despite, there was a stepwise increase in ECV values across troponin tertiles, and the modest differences are consistent with differences found in another study. It is also important to notice that to quantify T1 values with current techniques, a region of interest is drawn in a ventricular slice. This specific region is a sample, and its alterations may not correspond to the whole left ventricle characteristics, nevertheless, MASS V study included patients with multivessel disease who have diffuse ischemia and probably diffuse tissue alterations.Another possible criticism is that ECV values determined in our stable CAD patients were similar to those found in normal individuals in other studies. Reported values of ECV in normal myocardium are in the range of 24% to 28%,Finally, because we have only a single troponin I measurement performed before revascularization, we cannot be certain that some individuals did not have acute, rather than chronic cTnI elevation, nevertheless none of the patients had symptoms of recent onset, rapidly progressive or unstable angina.This is a cross-sectional study that has generated a hypothesis about scattered areas of myocardial injury and troponin release in stable CAD patients. As is well known, an inherent limitation of this type of study is that it cannot provide definitive information about cause-and-effect relationships. Additional studies with a larger sample of patients are required to confirm these findings.In this sample of patients, we found an independent association between troponin concentrations, native T1 and ventricular mass index and a correlation with CMR T1 mapping parameters, native T1 and ECV. These new findings indicate that focal areas of edema or fibrosis scattered in normal myocardium might be the anatomical substrate for chronic troponin release in stable coronary artery disease patients.Conceptualization: Carlos A W Segre, Célia Maria Cassaro Strunz, Whady Hueb.Data curation: Antonildes N Assunção Junior, Cesar Higa Nomura, Desiderio Favarato, Alexandre V Villa, José Rodrigues Parga Filho.Formal analysis: Carlos A W Segre, Antonildes N Assunção Junior, Cesar Higa Nomura, Desiderio Favarato, Célia Maria Cassaro Strunz, Alexandre V Villa, José Rodrigues Parga Filho.Funding acquisition: Whady Hueb.Methodology: Carlos A W Segre, Célia Maria Cassaro Strunz.Supervision: James A De Lemos, Whady Hueb.Writing – original draft: Carlos A W Segre.Writing – review & editing: Carlos A W Segre, James A De Lemos, Desiderio Favarato, Paulo Cury Rezende, Whady Hueb, Jose A F Ramires, Roberto Kalil Filho, Carlos Vicente Serrano Junior.


Patients with chronic kidney disease (CKD) have a higher prevalence of peripheral arterial disease (PAD), and endothelial progenitor cells (EPCs) play a pivotal role. We examined the impact of granulocyte colony-stimulating factor (G-CSF) on EPC function in response to tissue ischemia. Eight-week-old male C57BL/6J male mice were divided into sham operation and subtotal nephrectomy (SNx) groups, received hindlimb ischemic operation after seven weeks, then randomly received G-CSF or PBS intervention for four weeks with weekly follow-ups. SNx mice had significantly reduced limb reperfusion, decreased plasma EPC mobilization, and impaired angiogenesis in ischemic hindlimbs compared to the control group. However, G-CSF increased IL-10 and reversed these adverse changes. Additionally, ischemia-associated protein expressions, including IL-10, phospho-STAT3, VEGF, and phospho-eNOS, were significantly downregulated in the ischemic hindlimbs of SNx mice versus control, but these trends were reversed by G-CSF. Furthermore, in cultured EPCs, G-CSF significantly attenuated the decrease in EPC function initiated by indoxyl sulfate through IL-10. Overall, we discovered that G-CSF can improve EPC angiogenic function through a hypoxia/IL-10 signaling cascade and impede neovascular growth in response to ischemia of SNx mice. Our results highlight G-CSF’s potential to restore angiogenesis in CKD patients with PAD via EPC-based methods. Peripheral arterial disease (PAD) is a disorder characterized by the occlusion of arteries outside of the heart, most commonly in the legs. Patients with chronic kidney disease (CKD) or end stage renal disease (ESRD) who have undergone dialysis are at enhanced risk of PAD occurrence compared to the general population ,2. The oThe risk factors for PAD in CKD patients are not yet completely understood. However, it is probable that both conventional risk factors and those associated with CKD play a role in the development of this condition. Patients with CKD frequently experience reduced capacity for the formation of collateral vessels in ischemic tissues, which may further increase their risk of PAD. To better understand this phenomenon, we conducted studies using an animal model of CKD by performing subtotal nephrectomy (SNx) and then mimicking PAD by inducing hindlimb ischemia (HI) through surgery ,9. Our pGranulocyte colony-stimulating factor (G-CSF) is a cytokine that plays a crucial role in hematopoietic stem cell development and mobilization. Its therapeutic application involves the promotion of granulocyte production. For instance, recombinant human G-CSF is utilized in the treatment of patients with severe neutropenia during myelosuppressive therapy and bone marrow transplantation (BMT) . AdditioG-CSF was provided by UBI pharma Inc. . Fluorescein isothiocyanate (FITC) anti-mouse Sca-1 and phycoerythrin (PE) anti-mouse Flk-1 antibodies were purchased from eBioscience . Isotype-identical antibodies were obtained from Becton Dickinson . The antibodies listed below were purchased from Santa Cruz Biotechnology : rabbit anti-eNOS, anti-p-eNOS, mouse anti-VEGF, anti-α-SMA, and anti-β-actin. Rabbit antibodies for STAT3 and p-STAT3 were obtained from Cell Signaling Technology . Mouse antibodies for IL-10 were purchased from Serotec . Rat anti-CD31 was obtained from BD Pharmingen . Phenylmethylsulfonyl fluoride (PMSF) protease inhibitor, Histopaque-1077 (density: 1.077 g/mL), and 2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich . Endothelial growth medium (EGM-2 MV) and growth factors for EPC culture were obtained from Lonza .All animal experimental procedures were conducted in compliance with Animal Research Reporting of In Vivo Experiments (ARRIVE) guidelines and followed the protocols approved by the Institutional Animal Care and Use Committee (IACUC) of National Yang Ming Chiao Tung University ; the IACUC approval number is 1050621. A total of 33 male C57BL/6JNarl mice were purchased from the National Laboratory Animal Center, National Science Council . Experimental animals were accommodated in controlled environments with restricted access and maintained on a regular diet, adhering to a 12 h light and 12 h dark cycle.At 8 weeks old, 33 mice were allocated into three groups for the study: 7 mice for sham operation, 13 for SNx  with no treatment, and 13 for SNx treated with G-CSF. An intraperitoneal anesthesia consisting of 100 mg/kg of ketamine and 10 mg/kg of xylazine was administered prior to surgery. The SNx procedure involved initially excising both poles of the right kidney, followed by the complete removal of the left kidney after a one-week interval, which was carried out according to a previously described method [Seven weeks after SNx, the induction of hindlimb ischemia (HI) surgery involved the removal of the right femoral artery, following the established method described earlier . Prior tBlood samples were collected seven weeks after the SNx surgery and at the end of experiment to assess renal function and IS concentration. Serum was obtained from whole blood samples by centrifugation at 2500 rpm for 15 min, followed by careful collection of the supernatant. The resulting serum was then stored at −80 °C until measurement. The concentrations of blood urea nitrogen (BUN) and serum creatinine (Cre) were measured by an autoanalyzer . The concentration of plasma IS was determined using fluorescence-based high-performance liquid chromatography (HPLC). To prepare the samples, 50 mL of serum was combined with 150 mL of methanol to induce protein precipitation. After thorough mixing and subsequent centrifugation, the resulting supernatant (20 mL) was subjected to analysis. The separation of samples was accomplished using a Luna phenyl-hexyl column . The mobile phase consisted of 10 mM phosphate buffer with 5 mM tetra-n-butyl ammonium hydrogen sulfate and acetonitrile (3:1), and the flow rate was 1.0 mL/min. For quantification, the excitation wavelength was 280 nm, and a 346 cutoff filter was used to measure emission.A laser Doppler perfusion imager system  was utilized to measure the blood flow ratio between the ischemic limb (right) and the nonischemic limb (left) . FollowiBefore and 2 days after HI surgery, a fluorescence-activated cell sorting FACSCalibur flow cytometer  was used to assess EPC mobilization . A totalBlood samples were collected before animals were sacrificed to assess cytokine concentration. The IL-10 concentration in the mice serum was determined using a commercially available enzyme-linked immunosorbent assay kit  according to the manufacturer’s instructions.2O2. After blocking with 1% BSA for 1 h, the samples were incubated with anti-CD31 or α–smooth muscle actin (α-SMA) antibodies overnight at 4 °C, and then incubated for 2 h with an HRP (horseradish peroxidase)-conjugated secondary antibody at 37 °C. The immunoreactions were visualized using a TE2000-U fluorescence microscope . The number of CD31 or α-SMA positive cells were counted in 10 randomly selected high-power fields.After the animals were sacrificed, the thigh muscles of their hindlimbs were dissected. Harvested muscle tissues were fixed with 4% paraformaldehyde for one day and then dehydrated and embedded in paraffin. Tissue blocks were cut into 5 mm sections, then deparaffinized and incubated for 10 min with 3% HTM-P membrane . After blocking with 5% skim milk for a duration of 1 h, the membranes were sequentially incubated with primary antibodies and then with secondary antibodies. Protein bands were detected by using an enhanced chemiluminescence kit  and quantified with ImageQuant 5.2 software .Tissues were lysed with PBS containing 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 1 μg/mL leupeptin, 10 μg/mL aprotinin, 1 mM PMSF protease inhibitor, and phosphatase cocktail on ice. Following sonication, the tissue extracts were subjected to centrifugation at 12,000 times the force of gravity for 5 min at 4 °C. The resulting supernatants were collected as tissue lysates. To determine the protein concentrations, the Bradford protein-binding assay was employed. Aliquots containing 50 μg of protein from the lysates were separated using 8% SDS-PAGE and subsequently transferred onto an ImmobilonThe study about human EPCs was approved by the Ethics Committee of the Taipei Veterans General Hospital  with IRB number 2016-08-002AC. Informed written consent was obtained from the participants. Human EPCs were isolated from human peripheral blood and expanded in culture as described previously . The flu4) were placed in 96-well plates and then stimulated with G-CSF  for 24 h at 37 °C. We used a normal culture medium without G-CSF as the control, which produced a 100% survival rate. The cell viability of ECFCs was determined by measuring absorbance at 540 nm and is reported relative to the control.The cytotoxicity of G-CSF on ECFCs was assessed using a MTT assay. Isolated human ECFCs (2 × 104) were added in the upper chambers of trans-well plates (24-well) with polycarbonate membrane (8 mm pores). After 24 h of incubation, washed membranes were stained with Hematoxylin for 5 min, and then were mounted on a slide to observe under an inverted light microscope. The extent of ECFC migration was determined by counting the migrated cells in six randomly selected microscopic fields at a magnification of 100×.To assess the migratory capability of ECFCs, a modified Boyden chamber assay  was employed . Isolate4 cells/well into each matrigel-coated well in 100 μL of culture medium. After being cultured for 4 hrs, tube formation was examined under an inverted light microscope at a magnification of 100×. The number of branch points was assessed using image analysis for tube formation  in four randomly chosen microscopic fields. EBM-2 served as the culture medium for both assays.The in vitro tube formation of ECFCs was evaluated using an Angiogenesis Assay Kit  . The 96-p < 0.05.The data presented represent the mean ± SEM values obtained from 4–13 independent experiments. The Mann–Whitney test was employed to compare two independent groups, while the Kruskal–Wallis test, followed by the Bonferroni post hoc test, was utilized for comparisons among multiple groups. Statistical analysis was conducted using SPSS software version 18.0 . Statistical significance was determined at Mice were allocated into three groups, including sham operation (Sham), SNx, and SNx administrated with G-CSF (SNx + G-CSF) A. To conLaser Doppler imaging was employed to measure the hindlimb ratio of blood flow in both ischemic and non-ischemic limbs before and at various time points following the HI procedure A. All th+/Flk-1+ cells at baseline and two days following the hindlimb ischemia procedure. The results revealed that the G-CSF group exhibited significantly higher levels of EPCs compared to the sham mice and SNx mice, but no significant difference was observed between the sham mice and SNx mice (We investigated whether EPC mobilization played a role in the protective effect of G-CSF in our CKD mice model. To assess this, we assessed the levels of Sca-1SNx mice B. Since SNx mice C. Based + capillaries (+ small arteries (To evaluate the impact of G-CSF on neovascularization, we examined the density of blood vessels in the ischemic hindlimbs four weeks after the ischemic surgery. The findings disclosed that the intensity of CD31illaries A,B and αarteries A,C were At the four-week mark following the hindlimb ischemic surgery, we employed western blotting to quantify the levels of p-eNOS, IL-10, p-STAT3, and VEGF in the muscles of the ischemic limbs . Our finIn our study, we employed the MTT assay to examine how G-CSF affects ECFCs, as presented in Peripheral arterial disease poses a significant clinical challenge for patients with CKD. Lifestyle modifications, such as regular exercise, smoking cessation, and weight management, are among the strategies that can assist in managing PAD in these patients. Additionally, medications like statins, antiplatelet agents, and angiotensin-converting enzyme inhibitors may be employed to manage PAD and decrease the risk of cardiovascular events . In certThis present study shows that G-CSF reverses IS-induced inhibition of angiogenesis. We also examined the mechanism underlying this effect. G-CSF is a well-known myeloid growth factor, and previous studies have assessed the use of G-CSF as a prophylactic measure during chemotherapy-induced neutropenia, for retreatment after prior cycles of neutropenic fever, and to reduce the duration of chemotherapy-related neutropenia in afebrile patients . Due to Neovascularization is triggered by oxygen deficiency, prompting ischemic cells to release pro-angiogenic factors that attract nearby endothelial cells and stimulate cell proliferation ,24. ThesOur study showed that G-CSF significantly restored blood reperfusion after HI surgery, and there was no statistically significant disparity in hindlimb blood flow observed between the SNx + G-CSF group and the sham surgery group. These findings confirm that G-CSF promotes angiogenesis in the presence of CKD. As G-CSF is recognized for its ability to induce cell mobilization, we additionally quantified the number of circulating EPCs following HI surgery. The data demonstrate a significant increase in the number of EPCs in the peripheral blood at two days following the HI surgery with the administration of G-CSF.Ischemia upregulates HIF-1α, which then induces VEGF, leading to increased angiogenesis. IL-10 is also a pro-angiogenic factor, and previous research indicated that it promotes VEGF by increasing STAT-3 phosphorylation ,35. ThesIn conclusion, our study utilizing a mouse model of neovascularization in the presence of CKD demonstrates elevated IS levels following subtotal nephrectomy, leading to impaired reperfusion of the ischemic limb. The administration of G-CSF resulted in an increased number of circulating EPCs in the peripheral blood and promoted signaling of the IL-10/p-STAT-3/VEGF/p-eNOS pathway in the ischemic limb. G-CSF also promoted angiogenesis of ECFCs. Consequently, this experiment’s originality and novelty stem from the utilization of this animal model, which furnishes evidence indicating that G-CSF has the potential to serve as a promising therapeutic agent for treating PAD in patients with CKD. Since our study has demonstrated the effectiveness of this treatment in animals, in the future, further human trials will contribute to the results of this foundational research.


Polygonum orientale L. (PO) has a beneficial effect on treatment of myocardial ischemia (MI), its mechanism remains unclear. This study aimed to explore the pharmacological mechanism of PO against MI through MAPK signaling pathways. Firstly, the therapeutic effect of PO was evaluated for treatment of MI mice. Using Western blot and immunohistochemistry, the influence of PO on MAPK signaling pathways and cell apoptosis was investigated. Subsequently, one key pathway (ERK) of MAPK signaling pathways was screened out, on which PO posed the most obvious impact. Finally, an inhibitor of ERK1/2 was utilized to further verify the regulatory effect of PO on the MAPK/ERK signaling pathway. It was found that PO could reduce the elevation of the ST segment; injury of heart tissue; the activity of LDH, CK, NOS, cNOS and iNOS and the levels of NO, BNP, TNF-α and IL-6. It is notable that PO could significantly modulate the protein content of p-ERK/ERK in mice suffering from MI but hardly had an effect on p-JNK/JNK and p-p38/p38. Additionally, the expressions of bax, caspase3 and caspase9 were inhibited in heart tissue in the PO-treated group. To evaluate whether ERK1/2 inhibitor (PD98059) could block the effect of PO on treatment of MI, both PO and PD98059 were given to mice with MI. It was discovered that the inhibitor indeed could significantly reverse the regulatory effects of PO on the above indicators, indicating that PO could regulate p-ERK/ERK. This study provides experimental evidence that PO extenuates MI injury, cardiomyocyte apoptosis and inflammation by activating the MAPK/ERK signaling pathway.Although  Myocardial ischemia (MI), one of the common cardiovascular diseases with high mortality and morbidity worldwide, results from coronary atherosclerosis or blockage of myocardial blood supply and oxygen supply ,2. IncluPolygonum orientale L. (PO), a Chinese medicine derived from the family Polygonum, has an obvious effect on treating such diseases as coronary heart disease, chest tightness and shortness of breath [China has a long and distinguished history of traditional Chinese medicine (TCM) for treatment or prevention of diseases, dating back thousands of years . Polygonf breath . In prevf breath ,8,9, butf breath , also plf breath . Extracef breath . ERK1/2 f breath ,13. It hf breath . Moreovef breath ,15,16. Of breath ,18,19. HN-trans-feruloyltyramine and N-p-trans-coumaroyltyramine in PO extract.The base peak chromatograms (BPCs) of PO extract and standard solutions are displayed in p < 0.001), indicating that the MI model was established successfully. However, the ST segment was significantly decreased in PO and Danshen dripping pills (DS) groups (p < 0.05 or p < 0.01), showing that PO could significantly improve the elevation of the ST segment caused by MI.As showed in p < 0.01 or p < 0.001), while pretreatment with PO and DS significantly inhibited the activities of LDH and CK  (p < 0.05 or p < 0.001), while the activity of inducible NOS (iNOS) and the expression of IL-6 were higher (p < 0.001). Nevertheless, both PO and DS were able to observably ameliorate these indicators . These results reveal that PO could improve the function of regulating coronary artery contraction and relaxation and decrease inflammatory reaction.Compared with the sham group, the activities of LDH and CK in serum were markedly enhanced in the MI group (< 0.001) A,B, suggp < 0.01 or p < 0.001), but there appeared a significant decrease in bax and caspase 3 relative to the MI group (p < 0.001 or p < 0.001)  . These rp < 0.05), but the expression of p-ERK/ERK significantly increased (p < 0.01) in mice after administration of PO , while its level in the PO group was significantly higher than that in the MI group (p < 0.05). Nevertheless, the p-ERK level in the PO–PD (PO–PD98059) group was significantly lower than in the PO group (p < 0.05), indicating that the effect of PO on activation of ERK1/2 was blocked by PD98059 in MI mice . In the MI group, the activities of NOS, cNOS and NO level significantly decreased , and the contents of BNP, IL-6 and TNF-α significantly increased (p < 0.001) relative to the sham group. In the PO-treated group, however, PO significantly adjusted the changes of the ST segment, myocardial degeneration and necrosis as well as LDH, CK, iNOS, NOS, cNOS, NO, BNP, IL-6 and TNF-α . Compared to the PO group, the improvements conferred by PO disappeared in the PO–PD group, suggesting that PO indeed could regulate the ERK1/2 signaling pathway to decrease MI injury, cardiomyocyte apoptosis and inflammation.The expressions of ERK1/2 and p-ERK1/2 were detected using Western blot and immunohistochemistry. Compared with the sham group, the level of p-ERK decreased in the MI group , while PO could make them significantly lower (p < 0.001 or p < 0.01) in the PO group. However, the expressions of casepase3, caspase9 and bax in the PO–PD group were significantly higher (p < 0.05 or p < 0.01) than that in the PO group, indicating that PO indeed could activate the ERK1/2 signaling pathway to reduce the apoptosis induced by MI.The ERK1/2 signaling pathway is involved in the process of cell growth and differentiation in MI . On the PO, a Chinese medicine with good medicinal value derived from family Polygonum, is commonly used in rheumatoid arthritis, CAD, stomachache and other diseases . In prevIn this study, a range of indicators were tested to investigate the efficacy of PO on improvement of MI, including the change of the electrocardiogram; the activities of LDH, CK, NOS, iNOS and cNOS and the levels of NO and apoptotic proteins in mice. The effect of PO on the key proteins p38, JNK and ERK1/2 in the MAPK signaling pathway was analyzed by Western blot. Interestingly, the results showed that there were no significant changes in the regulation of p38 and JNK during the process of PO treating MI, but PO could significantly activate the ERK1/2 signaling pathway. Furthermore, it was confirmed that this activation of ERK1/2 could be impeded by the inhibitor PD98059. There was evidence showing that PO might increase the activities of NOS and cNOS and the level of NO; decrease the activities of iNOS, LDH and CK and the level of BNP; inhibit the content of inflammation-related factors IL-6 and TNF-α and reduce the expression of apoptosis-related proteins. Accordingly, this influence could be blocked by PD98059, suggesting that the ERK1/2 signaling pathway was involved in the process during which PO exerted therapeutic effect on MI.Extracellular signal-regulated kinases (ERKs), specifically ERK1 and ERK2, are key enzymes that transmit signals from surface receptors to the nucleus . The ERKIt is widely acknowledged that MI can give rise to an increase in oxygen free radicals destroying the balance between oxidants and antioxidants, resulting in myocardial injury and the release of such myocardial enzymes as LDH and CK ,24. BNP,It has been reported that activation of the ERK signaling pathway can reduce the inflammatory response in MI injury . InflammNO, a main vasodilator in the body and an important endogenous myocardial protective factor, plays an important role in regulating the cardiovascular system . EndogenPolygonum orientale flower extract can significantly increase the protein phosphorylation level of ERK1/2 protein in hypoxia-reoxygenation injury of H9c2 cardiomyocytes and decrease caspase3 and bax to inhibit cardiomyocyte apoptosis [Of great importance is apoptosis among mechanisms of myocardial ischemic injury . In MI ipoptosis ,43. The This study preliminarily confirmed that PO can reduce myocardial pathological injury; decrease the levels of IL-6, BNP and TNF-α in serum; increase the activities of NOS and cNOS and the level of NO and decrease the levels of iNOS, caspase3, caspase9 and bax by activating the ERK1/2 signaling pathway in MI mice.Adult KM mice (25–30 g) were procured from the animal center of Guizhou Medical University . They were housed adaptively for 1 week in an animal house at a suitable temperature (18–25 °C) and humidity (50–70%) and had free access to water and food. The study protocol was approved by the Animal Ethics Committee of Guizhou Medical University Animal Center .N-trans-feruloyltyramine (AF20060301), orientin (lot no. AF9052413), isoorientin (lot no. AF20051551), vitexin (lot no. AF8111891) and N-p-trans-coumaroyltyramine (lot no. AF20060304) were obtained from Chengdu Alfa Biotechnology Co., Ltd. . Quercitrin (lot no. wkq18041101) was provided by Sichuan Weikeqi Biological Technology Co., Ltd. , the purity of which was more than 98%.Enzyme-linked immunosorbent assay (ELISA) kits for determination of IL-6 (KE10007) and TNF-α (KE10002) were provided by Proteintech Group, Inc. . BNP(H166), NOS(A014-1), LDH(A020-2), CK (A032-1) and NO (A013-2-1) test kits were purchased from Jiancheng Bioengineering institute . PD98059 (lot no. MKCN2664) was obtained from Sigma Aldrich . Anti-caspase3 (9662S), anti-caspase9 (95047) and bax (27727) were purchased from Cell Signaling Technology . Anti-JNK (ab179461), anti-phosphorylation JNK (ab124956), anti-p38 (ab170099), anti-phosphorylation p38 (ab195049), anti-ERK1/2 (ab184699), anti-phosphorylation ERK1/2 (ab214036) and anti-GAPDH (ab181603) were obtained from Abcam . The BCA and enhanced chemiluminescence kits were bought from Dalian Meilun Biotechnology Co., Ltd. . Tribromoethanol (C11707118) and tert-pentyl (F2004116) alcohol were supplied by Shanghai Macklin Biochemical Co., Ltd. and Shanghai Aladdin Bio-Chem Technology Co., Ltd., respectively. Protocatechuic acid (lot no. AF6121206), N-trans-feruloyltyramine and N-p-trans-coumaroyltyramine) of PO were 29.49, 115.26, 151.58, 24.09, 2.03, 79.79, 5.98 and 2.64 mg/g, respectively.The extract of PO was prepared through referring to the previous experimental method . BrieflyPO extract (about 10 mg) was weighed and dissolved in 10 mL of 50% aqueous methanol, and then the solution was filtered through a 0.22 μm membrane, the filtrate of which was collected for identification of chemical profiles of PO using UHPLC–Q-Exactive Orbitrap Plus HRMS.The reference compounds were accurately weighed and dissolved in 10 mL methanol to obtain the stock solution. Mixed reference solution (2–10 μg/mL) was prepared by diluting each stock solution with 50% methanol.The analysis was performed on a Vanquish horizon UHPLC coupled with Q-Exactive Plus HRMS Spectrometer . The Q-Exactive Plus HRMS Spectrometer equipped with an electrospray ionization (HESI) source was employed for MS in both positive and negative modes. The MS conditions were as follows: spray voltage 3.5 kV for positive ion mode and 2.5 kV for negative ion mode, capillary temperature 320 °C, ion source sheath gas flow rate 35 arb, aux gas flow rate 10 arb, probe heater temperature 350 °C and the stepped NCE was set to 20, 40 and 60 V. The scan ranges of a full MS scan with a resolution of 70,000 were set to m/z 100–1500, and dd-MS2 at a resolution of 17,500 was recorded from 100 to 1500 m/z.v/v) and B (acetonitrile), at the flow rate of 0.3 mL/min and with the injection volume of 2 μL. The gradient elution procedure was as follows: 6% B from 0 to 0.5 min, 6–8% B from 0.5 to 2 min, 8–13.5% B from 2 to 5 min, 13.5% B from 5 to 21 min, 13.5–30% B from 21 to 35 min, 30–80% B from 35 to 38 min and 80–95% B from 38 to 39 min.Chromatographic separation was performed on a Hypersil gold column  at 40 °C with mobile phase A  with tert-pentyl alcohol. According to a method reported in references to establish the MI model , brieflyn = 6), namely a sham group, myocardial ischemia group (MI), PO group  and Danshen dripping pills  group. Danshen dripping pills are clinically used to treat coronary artery disease [The experimental protocol was divided into two parts. The first was designed to explore the effect of PO on MI in mice and the regulation of the MAPK signaling pathway. Mice were divided into four groups : the sham group, myocardial ischemia group (MI), PO group (PO) and PO–PD98059 group (PO–PD). The mice in the PO group were orally administered PO extract (4 g/kg). For the PO–PD group, before administering PO extract for 30 min, mice were intraperitoneally injected with PD98059 (1 mg/kg) based on Reference [The second was intended to investigate and verify the role of ERK1/2 signaling in the process during which PO exerted a protective effect on MI. The mice were divided into four groups  to take the total protein, concentration of which was measured by a BCA kit. Firstly, the total proteins were separated using 10% SDS-PAGE, then transferred to PVDF membranes. After blocking with efficient Western blocking fluid for 15 min, the membranes were incubated with the following specific primary antibodies overnight at 4 °C: anti-p38 (1:5000), anti-p-p38 (1:1000), anti-ERK1/2 (1:5000), anti-p-ERK1/2 (1:1000), anti-JNK (1:1000), anti-p-JNK (1:5000), anti-GAPDH , anti-bax (1:1000), anti-caspase3 (1:1000) and anti-caspase9 (1:1000). Subsequently, these membranes were incubated with adding HRP-conjugated secondary antibodies (1:5000) for 2 h at room temperature. Lastly, the membranes were visualized using a gel imaging system with an enhanced chemiluminescence kit and quantified using Quantity One software.Heart tissue was homogenized and centrifuged . The sections were incubated overnight at 4 °C with primary antibodies such as anti-ERK1/2 (1:500), anti-p-ERK1/2 (1:480) and PBS (a negative control). Then, sections were reacted with goat anti-rabbit or mouse HRP-conjugated secondary antibodies at 37 °C for 30 min. Diaminobenzidine was used to visualize the reaction product. Finally, the sections were washed, then stained with hematoxylin and finally visualized under a microscope.p < 0.05 was considered statistically significant.Data were analyzed using GraphPad Prism software 7 and expressed as mean ±SD. One-way analysis of variance (ANOVA) was used for comparison when more than two groups were compared. In summary, the mechanism of PO for treatment of MI may be that PO activates the MAPK/ERK signaling pathway, downregulating the expression of such apoptosis-related proteins as bax, caspase3 and caspase9; improving myocardial inflammation; increasing the NOS activity and NO level in serum to dilate blood vessels and increase the myocardial blood flow and oxygen supply.


Tumor thrombus occurs when tumor cells extend into a blood vessel. An estimated 10% of kidney cancer cases are complicated by tumor thrombus, often invading the renal vein with extension to the inferior vena cava. Up to 1% have tumor cells extending to the heart. The standard of care for these patients is surgical removal of the kidney tumor and the tumor thrombus. Research focuses on surgical techniques, imaging methods, and molecular markers for prognosis. The full benefit of anticoagulation remains controversial in these cases, considering unknown benefits and bleeding risk during tyrosine kinase inhibitor therapy. In this literature review, we summarize known data regarding the use of anticoagulation in the setting of kidney cancer and tumor thrombus.Tumor thrombus (TT) is a complication of renal cell carcinoma (RCC) for which favorable medical management remains undefined. While radical nephrectomy has been shown to increase overall survival in RCC patients, surgical interventions such as cardiopulmonary bypass (CPB) and deep hypothermic circulatory arrest (DHCA) utilized to perform TT resection carry high mortality rates. While it has been documented that RCC with TT is associated with venous thromboembolism (VTE) development, anticoagulation use in these patients remains controversial in clinical practice. Whether anticoagulation is associated with improved survival outcomes remains unclear. Furthermore, if anticoagulation is initiated, there is limited evidence for whether direct oral anticoagulants (DOACs), heparin, or warfarin serve as the most advantageous choice. While the combination of immunotherapy and tyrosine kinase inhibitors (TKIs) has been shown to improve the outcomes of RCC, the clinical benefits of this combination are not well studied prospectively in cases with TT. In this literature review, we explore the challenges of treating RCC-associated TT with special attention to anticoagulation. We provide a comprehensive overview of current surgical and medical approaches and summarize recent studies investigating anticoagulation in RCC patients undergoing surgery, targeted therapy, and/or immunotherapy. Our goal is to provide clinicians with updated clinical insight into anticoagulation for RCC-associated TT patients. Renal cell carcinoma (RCC) comprises 3% of all cancers . It is pClinical findings of RCC with TT are often insidious and nonspecific, such as palpable abdominal mass . AdditioThe Mayo Clinic has a defined grading system for TT extension, with a scale of grade 0–IV . HoweverCurrent National Comprehensive Cancer Network guidelines do not address anticoagulation in patients with RCC-associated TT, leaving the decision to initiate anticoagulation to the physician’s discretion. The use of anticoagulation with receptor tyrosine kinase (RTK) and multi-kinase inhibitors poses a bleeding risk and remains a clinical concern . As noveTumor thrombi are classified on a graded scale of 0–IV. Type 0 is limited to the renal vein, type I involves infiltration of the IVC less than 2 cm above the renal vein, and type II extends to the IVC below the hepatic veins. Type III is defined as the progression of TT up to the hepatic veins but below the diaphragm, and type IV extends above the diaphragm and includes the right cardiac atrium ,14. The While it is well documented that a complete lack of TT is associated with an improved prognosis, discrepancies have arisen regarding whether the total TT burden affects prognosis. Previous studies have demonstrated no significant difference in overall survival among patients with grade 0–IV TT ,10. SubsAdditional factors of TT composition are being studied to determine whether the components of the TT itself affect survival and prognosis. In a recent study conducted by Jia et al., the effect of TT consistency on RCC patient prognoses was assessed . This stA retrospective analysis was conducted by Liu et al. at Peking University Third Hospital, which involved patients with RCC and TT who were undergoing surgery . The purIt was postulated by the authors of this paper that a TT growing against the direction of venous return results in sluggish blood flow, causing platelets and red blood cells to accumulate at the distal end of the TT, thus prompting the formation of lengthy bland thrombus. The propensity of TT growing against venous return prompts consideration as to whether anticoagulation should be initiated in these patients. Further investigation into the directionality of TT growth is necessary to determine whether anticoagulation is warranted for patients with TT growing against the venous return. This study is the sole exploration of TT growth directionality that we could find. The complexity and location of a TT require thorough pre-surgical planning aided by imaging. If the TT is found to invade the IVC wall, resection of this area of the IVC is necessary. However, when the TT is large enough to fill the entire IVC lumen, it becomes especially challenging to determine whether the TT is invading the IVC wall or simply causing compression. On CT images, distinguishing between a TT and bland thrombus is difficult since they appear similar. A biopsy with pathologic analysis, which is often not feasible, would be necessary to differentiate whether the clot is a TT or thromboembolic .Magnetic resonance imaging (MRI) outperforms computed tomography (CT) imaging in distinguishing between TT and bland thrombi . A recenA study conducted by Sarswat et al. closely examined the role of FDG PET/CT in the diagnosis of benign versus malignant TT . Among 1Nephrectomy with thrombectomy is currently the standard in surgical care for patients with RCC and TT . CPB witStudies concerning the surgical management and outcomes of RCC with TT are hindered by two key factors: small sample sizes and extended time spans ranging from several years to decades . BecauseLaparoscopic approaches may provide a better safety profile to treat RCC with TT. Fourteen patients who underwent laparoscopic radical nephrectomy in the setting of RCC were retrospectively identified, four of whom had TT localized to IVC. It was deemed that for these four patients, laparoscopic nephrectomy with thrombectomy was safe and effective . A matchInvestigation of molecular methods to determine prognosis of RCC with TT is currently underway. Shi et al. performed a genetic analysis of clear cell RCC with TT, aiming to identify distinct molecular features associated with the development of TT . They esAdditionally, a series of microRNAs has been identified in patients with clear cell RCC with the TT extending to the IVC: miR-221, miR-126, and miR-2126. Expression levels of these three microRNAs have been shown to be deregulated in thrombosis and embolic events . This paA case report by Labbate et al. performed multichannel immunofluorescence on TT tissue and demonstrated that TT was characterized by infiltration of immune cells, including CD + 8T cells, FoxP3+ regulatory T cells, and Batf3+ dendritic cells . InteresElucidation of biomarkers could pave the way for the development of molecular subtypes of RCC with TT. Ideally, these biomarkers could provide prognostic tools to assess the risk of progression and response to treatment. Biomarkers known to be directly involved in thromboembolism could aid in determining whether the patient would benefit from anticoagulation. Further exploration into TT biomarkers is warranted, as are studies of the local TT microenvironment and epigenetic regulation.It remains debatable as to whether anticoagulation improves survival in cases of RCC with TT. A retrospective review of 153 patients with malignancy and TT treated with or without anticoagulation found no difference in survival between groups . It is wA recent study performed at Leiden University in the Netherlands evaluated the outcomes of anticoagulation in patients with RCC and TT . The decIt has been demonstrated that tyrosine kinase inhibitor (TKI) therapy improves overall survival in patients with RCC. TKIs are known to improve progression-free as well as overall survival. Interestingly, targeted therapies offer comparable benefits to patients over the age of 65 as they do to younger patients . HoweverCabozantinib, a drug that inhibits multiple tyrosine kinase receptors including VEGF, is approved for the treatment of RCC. Its efficacy was demonstrated in the CABOSUN and METEOR clinical trials. A multicenter study performed by Shayeb et al. examined the safety of cabozantinib and venous thromboembolism (VTE) formation. They found that DOACs may be safe for use with cabozantinib . A totalSeveral case reports and limited studies have explored the effects of immunotherapy on RCC with TT . In a prAn additional case of a 54-year-old female diagnosed with locally advanced clear cell RCC with level IV TT was initially determined to be unfit for surgery due to poor performance status . NeoadjuIn another case report, a 67-year-old male with grade IV TT and RCC was treated with dual checkpoint inhibitor immunotherapy with nivolumab and ipilimumab . FollowiA 71-year-old female, also with grade IV TT and RCC, received combined immunotherapy with nivolumab and ipilimumab. She experienced TT progression into the right atrium while undergoing treatment . TreatmeA study of five patients performed by Yoshida et al. further evaluated the effect of neoadjuvant immunotherapy on RCC with TT . Three pA single, multi-center, phase II study (NAXIVA) investigated neoadjuvant axitinib administration for eight weeks in patients with both metastatic and non-metastatic clear cell RCC and TT prior to resection. Out of twenty evaluable patients, seven experienced a reduction in TT level. Among those who underwent surgery, seven out of seventeen had less invasive procedures than initially planned . AdditioIt is notable that many of these patients treated with TKI and/or immunotherapy had subsequent downstaging of TT level. Efforts are currently underway to guide decision making for treatment options in this scenario, as demonstrated by Wu et al. The study was a retrospective analysis of tumor grade discrepancy between the primary tumor and TT in nonmetastatic clear cell RCC . They foPresurgical sunitinib has also been shown to reduce primary tumor size in patients with RCC, facilitating partial nephrectomy and preserved renal function in a clinical trial of 72 patients  Table 2Table 2. There are currently no ongoing randomized controlled trials comparing the safety and/or efficacy of anticoagulation in patients with RCC-associated TT. The limited size of study samples and the diverse nature of RCC cases with TT present challenges when attempting to establish broad guidelines for anticoagulation. It is clear that certain TKIs can interact with warfarin. Pazopanib, a multi-tyrosine kinase inhibitor approved to treat RCC, has been shown to prolong international normalized ratio (INR) and result in liver dysfunction, thus requiring close monitoring and even termination of both anticoagulation and pazopanib therapy . This haMultiple case reports documenting LMWH use in the setting of RCC with TT provide limited insight. For instance, a 70-year-old male with no past medical history was found to have anterior abdominal wall collateral vessels on physical examination and elevated inflammatory markers . Right aA case of a 60-year-old male with a history of 20-pound weight loss, night sweats, fevers, and back pain with palpable abdominal mass was found to have RCC with TT into IVC and right atrium . He had A review article exploring the management of IVC-localized TT suggests the potential need for anticoagulation and recommends LMWH as the preferred anticoagulant .A phase I study examined the outcomes of dalteparin with sunitinib use in patients with metastatic clear cell renal carcinoma . NotablyThe existing literature tends to focus on the safety of DOACs in patients with cancer-associated VTE rather than TT. Limited information is available regarding the use of DOACs with TT. The aforementioned cabozantinib study performed by Shayeb et al. suggests that DOACs might be as safe as LMWH when used concurrently with cabozantinib, as there were no significant differences in major bleeding events among the no anticoagulant, DOAC, and LMWH groups .Treatment of RCC with TT remains understudied and warrants further prospective evaluation. Limited studies suggest that neoadjuvant treatment with immunotherapy can shrink TT, potentially offering an additional benefit in pre-surgical settings. Concurrent treatment with TKIs has also demonstrated a reduction in TT size. However, administering TKIs with anticoagulation poses a greater risk of bleeding. There is no well-documented overall survival benefit for patients with RCC-associated TT. Patients treated with anticoagulation can experience major bleeding events that can be life-threatening. Further research and subgroup analysis are necessary to determine which patients would clinically benefit the most from anticoagulation administered for TT treatment.


In this work, femtosecond laser pulses at high repetition rates were used to fabricate unique microstructures on the surface of Ti6Al4V. We investigated the influence of pulse overlap and laser repetition rates on structure formation. Laser texturing with a high degree of overlap resulted in melting of the material, leading to the formation of specific microstructures that can be used as cavities for drug delivery. The reason for melt formation is attributed to local heat accumulation at high repetition rates. Such structures can be fabricated on materials with low thermal conductivity, which prevent heat dissipation into the bulk of the material. The heat accumulation effect has also been demonstrated on steel, which also has low thermal conductivity. One of the biggest challenges in manufacturing implants is to create a bio-interactive surface that bacteria cannot attach to, while at the same time producing an implant with strong mechanical properties. Titanium and titanium alloys are known for their high corrosion resistance, biocompatibility, low modulus of elasticity, and strength . Due to In recent years, laser texturing has become a common method of surface modification in medical technology ,24,25. CAfter implantation, the patient’s immune system is weak, which allows bacteria to spread rapidly. Hence, the suppression of a bacterial attack is important during the first hours and days after implantation. Pathogen microbes causing infections on the implant area are delivered through the bloodstream, for example, from inflammation of a tooth leading to the septic implant loosening . For thiIn this paper, we show that structures fabricated at a high degree of fs-laser pulse overlap and high repetition rates results in a unique form of structures. Ti alloy implants that have micro-cavities on their surface can be used in the implant industry. Micro-cavities can be filled with drugs before implantation, so that the drugs act locally after implantation and prevent the adhesion of bacteria. The formation of such structures is a result of a complex material interaction with an incident laser beam that is dependent on electron–phonon (e–ph) coupling constant, thermal conductivity, and other physical properties of the material, wavelength, and polarization of the incident light, machining environment, and laser parameters, such as the laser fluence, laser absorptivity, and degree of pulse overlap . SeveralThe investigations were performed on Ti6Al4V samples with a dimension of 2 cm × 2 cm × 1 cm (L × W × H) with an average surface roughness Sa of approximately 0.95 ± 0.03 µm (according to DIN EN ISO 4287). Ti6Al4V plates were purchased from ARA T Advance GmbH  and correspond to the requirements of ASTM F136. In the second row of experiments, pure copper  and stainless steel  samples were also laser textured. Prior to laser texturing, all samples were cleaned in an ultrasonic bath  in acetone for 5 min. Drying was applied with dust-free wipes and compressed air.2 ~ 1.4) with a maximum pulse energy of 20 µJ. The laser beam is focused on the surface of the target with a two-axis scan head-type Fiber Rhino  equipped with a F-theta lens with a focal length of 160 mm, leading to a circular focus diameter of fd = 30 µm. The laser texturing parameters were varied over a wide range to yield various microstructures and to investigate their effect.Laser texturing of samples was performed by a Yb-doped femtosecond fiber laser YLPF-10-500-10-R . The laser system delivers a beam at a wavelength of 1030 nm with pulses in the range of 120–650 fs and operates at a pulse repetition rate between 150 kHz and 1 MHz. Spatial intensity distribution of the beam is nearly equal to a spatial Gaussian profile (beam parameter product MPO) was calculated from Equation (1):POs were achieved by an adjustment of the scanning velocity according to Equation (1). The used processing parameters are listed in The laser pulse overlap . The profile of the resulting structures was measured with a confocal laser scanning microscope .2 during these experiments on Ti alloy.When comparing the structures obtained at repetition rates of 150 kHz, 500 kHz, and 1 MHz, note that the only difference between the applied laser parameters is the time interval between successive laser pulses, which results from the applied repetition rate. The time interval between pulses at repetition rates of 150 kHz, 500 kHz, and 1 MHz accounts to 6.6 µs, 2 µs, and 1 µs, respectively. These time intervals are an important factor that allow us to demonstrate the effect of heat accumulation as a function of the applied repetition rate. In turn, the laser fluence was kept constant at 1.4 J/cmPO = 98.80 and PO = 99.10 with different repetition rates show that a slight change in PO can result in a completely different type of structure. The textured surfaces at PO = 99.80 with repetition rates of 150 kHz and 500 kHz are relatively similar, although the surfaces textured with a repetition rate of 500 kHz have small particles that resemble redeposition particles. The surfaces textured with the same PO at 1 MHz again differ slightly from those textured at 150 kHz and 500 kHz. A SEM image shows that the textured lines have micro-holes along a grid line, and the textured area looks smoother compared to its original surface. When comparing PO = 98.80% and PO = 99.10%, no changes are observed on surfaces textured at a repetition rate of 150 kHz. Surfaces textured with a repetition rate of 500 kHz and 1 MHz, by contrast, differ significantly. Surfaces textured at f = 500 kHz yielded solidified bump-like structures with periodic micro-holes, while the surface textured at f = 1 MHz resembles shallow trenches with micro-holes that were not as frequent as those formed at f = 500 kHz. A further increase in PO to 99.50 at f = 150 kHz showed increased material removal leading to the formation of trenches on the Ti alloy surface, and the surface fell below the original surface level as shown in f = 500 kHz resulted in enlargement and coarsening of the structures, while the surface textured at f = 1 MHz led to the formation of deeper trenches. Comparison of representative SEM images of structures fabricated at PO = 99.10 and PO = 99.50 at f = 500 kHz shows that increasing PO leads to increased formation of molten material with subsequent resolidification. When comparing surfaces textured at PO = 99.10 and PO = 99.50 at f = 1 MHz, it can be seen that increased PO leads to increased material removal as the number of laser pulses per area increases with increasing PO. When comparing trenches made at PO = 99.50 with different repetition rates, the edges of the trenches made at f = 1 MHz look smooth and resolidified, while those made at f = 150 kHz are sharp-edged, indicating clear material removal. Increasing PO to 99.58 at f = 500 kHz showed no difference between structures made with PO = 99.50, while trenches fabricated with f = 150 kHz became deeper. In turn, the trenches at f = 1 MHz became deeper, and a more molten material solidified at their edges. SEM images of structures made with PO = 99.66 and PO = 99.75 show that the trenches became even deeper at f = 150 kHz, while at f = 500 kHz no visible change in the structures is observed. In the case of f = 1 MHz at PO = 99.66, no resolidified material was observed at the edges of the tranches, while at PO = 99.75 the trenches became deeper, and the amount of resolidified material at the edges increased. It appears that at high PO levels and high repetition rates, the formation of excess molten material leads to the formation of unique structures, and the molten material is driven out of the inside of the trenches during laser texturing. Even if the applied fluence is the same, the time interval between pulses seems to play an important role in the formation of such structures. To the best of our knowledge, such structures are reported for the first time. A further increase in PO resulted in the disappearance of the trenches when repetition rates of 150 kHz and 500 kHz were used. The reason for this could be a slow laser scan speed that results in a dense plasma plume, which does not allow the subsequent laser pulses to reach the surface of the sample. At f = 1 MHz and POs up to 99.86%, a larger amount of resolidified material accumulated at the edges of the trenches, and at PO = 99.90 and above, the trenches disappeared completely. It appears that at high repetition rates, the textured surface melts and resolidifies at the textured areas of the sample. Overall, the size and morphology of the structures depends not only on the fluence but also on the laser repetition rate. The main structure types to be generated are micro-holes and bump-like structures. It can be clearly observed that at higher repetition rates melt formation is more pronounced.From the comparison of the SEM images, it can be concluded that the applied repetition rate had a great influence on the structures formed, although the applied laser fluence and number of pulses were kept constant. For example, the structures fabricated at To summarize the evolution of surface topography during laser texturing, we performed confocal laser scanning microscopy (CLSM) measurements on textured samples. The results of the CLSM are shown in f = 1 MHz and PO = 99.90, with the black line representing the line scan profile at a representative position perpendicular to the fabricated laser trenches. The corresponding line scan profile is depicted below the CLSM image. Line scan measurements were conducted five times on each resulting structure at extreme values, i.e., at the lowest positions in the case of pure trenches, where no melt formation is observed, and at the highest positions, where the structures are a result of melt formation and resolidification. The local extreme values were measured in each line profile and are averaged for each PO value of the laser fabrication process. PO at a repetition rate of f = 150 kHz, f = 500 kHz, and f = 1 MHz. The x-axis indicates the degree of PO, while the y-axis provides information on the averaged extreme values relative to the original sample surface at a given PO. The red line in f = 500 kHz. At f = 150 kHz or f = 1 MHz, by contrast, the structured trenches become even deeper. The later transition of the trenches into the melt at f = 1 MHz could be due to the faster scan speed compared to f = 500 kHz, where heat dissipation into the bulk could play an important role in the formation of structures. Nevertheless, the transition to melt-induced structures also occurs at f = 1 MHz, and the height of the resulting structures is even larger than those induced at f = 500 kHz. This could indicate that the amount of melted material is larger at f = 1 MHz than at f = 500 kHz. Such behavior could be caused by shorter time intervals between laser pulses at f = 1 MHz, which could lead to local heat accumulation. When the PO was further increased, at f = 150 kHz, the trenches became even deeper and did not result in excessive melting of the material leading to the formation of preferred structures, as was the case at f = 500 kHz and f = 1 MHz. Nevertheless, a slight transition to melt-induced structures can be observed at PO = 99.86 at f = 150 kHz. However, a look at the SEM image at PO = 99.86  using ultrashort laser pulses. With an increasing PO and a repetition rate, various surface structures were obtained. The reason for the formation of such structures is attributed to local heat accumulation at high repetition rates, which leads to melting of the material during laser texturing.This paper presents a comparative study on laser surface texturing of Ti6Al4V at high repetition rates and a high degree of pulse overlap (In the case of Ti alloy and steel, the reason for the formation of melt is their strong e–ph coupling constant, which allows a fast energy transport of laser pulses into the material lattice, but heat dissipation is insufficient due to low thermal conductivity, by contrast, has a high thermal conductivity and is less prone to melting at high repetition rates because the heat dissipates faster into the surrounding bulk. These effects have to be taken into account for specific laser system and material combinations, especially for novel developments in high-repetition ultra-short pulse lasers. Limited investigations were able to show such phenomena . Melt foPO and repetition rates that allows us to choose an appropriate texturing regime with preferred structures on Ti alloy depending on the application. Based on this graph, it is possible to move from pure trenches without melting to those formed by melting of the material depending on the applied repetition rate. In principle, these structures can be used for drug delivery systems on the surface of Ti alloy using ultrashort laser pulses in the kHz and MHz regimes.Additionally, we presented a graph with In the future, our studies will focus on mechanical testing of textured Ti samples and the characterization of drug delivery properties of these structures.


This study investigated the relationship between the systemic immune inflammation index (SII) and catheter-related infections and their effects on prognosis in pediatric patients.A total of 56 pediatric patients diagnosed with ventriculoperitoneal (V-P) shunt infection between January 2017 and October 2019 were included. V-P shunt infection diagnosis was made based on clinical findings. All cerebrospinal fluid (CSF) samples were subjected to direct microscopic examination and culture. Protein, glucose, and sodium levels in CSF, CSF leukocytes, and hematological and biochemical parameters were measured.p = 0.046), and SII was significantly increased (p = 0.002) in non-coagulase-negative staphylococci.Fifty-six patients with growth in CSF culture were included in this study. 55.4% of the cases were female and 44.6% male. V-P shunt was detected in 82.1% of the cases and external ventricular drainage (EVD) catheter-related infection in 17.9%. The CSF/blood glucose ratio was significantly lower (Early and appropriate antibiotic therapy reduces morbidity and mortality in catheter-related infections. However, it is important to start empirical antibiotherapy until culture results are expected. Therefore, further research on the estimation of possible factors is needed. The central nervous system is protected by the blood–brain barrier against microbial invasion. However, the disruption of the blood–brain barrier could lead to infection . VentricStaphylococcus aureus, coagulase-negative staphylococci (CoNS), gram-negative bacteria, and anaerobic bacteria. Among these pathogens, anaerobic and gram-negative bacteria are generally known to cause benign and slow infections. In contrast, gram-positive bacteria cause more aggressive and rapid infections. Among the gram-positive bacteria, S. aureus is thought to cause more rapid-onset infections than CoNS  is a system used for intracranial pressure monitoring, CSF drainage in the occluded ventricular system, or the treatment of infected catheters. It is also an important cause of nosocomial meningitis. EVD-related infections have been known to have a rate of 9%–20% . CoNS, Sathogens .Platelet activation and leukocytosis occur as a result of the stimulation of the systemic immune reactions after catheter-related central nervous system infections. Increased inflammatory biomarkers have important effects on the severity of the disease, response to treatment, and prognosis in catheter-related central nervous system infections . RecentlIn recent years, SII has been shown to have a prognostic value in estimating different malignant diseases . HoweverPediatric patients with a diagnosis of V-P shunt or EVD catheter-related central nervous system infection confirmed by growth in CSF cultures between January 2017 and October 2019 were retrospectively included in this study. Patients’ age, gender, complete blood count at hospitalization, biochemistry, C-reactive protein (CRP) levels, SII, catheter type, clinical findings, laboratory characteristics, CSF findings , CSF culture results , length of hospital stay, treatments , and the final status of the patients were recorded in the follow-up forms. SII was calculated according to the following formula: SII = Platelet × Neutrophil/Lymphocyte. NLR was calculated according to the following formula: NLR = Neutrophil/Lymphocyte. PLR was calculated according to the following formula: PLR = Platelet/Lymphocyte. Patients with positive CSF culture and unavailable data were not included in this study.The diagnosis of catheter-related central nervous system infection was made based on the results of clinical findings and the CSF examination. And the diagnosis was confirmed by bacterial growth in the CSF culture. No antibiotic therapy was used to prevent infection. The same shunt systems were implanted in all study patients. A pediatric, ultrasmall, flat-bottomed, medium-pressure ventriculoperitoneal shunt systems were used . Empirical antibiotic therapy was started until the CSF culture results were obtained. Ceftriaxone was started for patients older than 1 month, and a combination of ampicillin and gentamicin was administered to newborns. After the CSF culture results were obtained , specific antibiotic therapy was started. CSF culture results were repeated after the patients were treated for an average of 2 weeks. Antibiotics were discontinued, continued, or changed according to the results of the second culture. It was thought that the patient was cleared of infection, with negative CSF culture results after antibiotic therapy and a decrease in white blood cell count in CSF.2 for 72 h. At the end of the incubation period, the VITEK 2 automated system  was used to identify the colonies with growth at the species level and to determine their antibiotic susceptibility. Antimicrobial susceptibility results were evaluated according to the current Clinical and Laboratory Standards Institute criteria (All CSF samples taken under sterile conditions and sent to the laboratory were directly subjected to microscopic examination (gram staining) and culture. The detection of the microorganism grown in culture using gram analysis greatly reduces the possibility of contamination. The CSF samples were first centrifuged. The upper fluid was used for biochemical examination, while the bottom sediment was used for microbiological examination. Gram staining was done on a part of the precipitate and examined microscopically. The rest of the colony was cultivated on chocolate agar, blood agar, and eosin methylene blue agar. The plates were incubated at 37 °C for 18–48 h, and the chocolate medium was incubated in an oven with 5% COcriteria . Our stup value of <0.05 was considered statistically significant.Statistical analysis was performed using the SPSS statistical program . All clinical parameters were analyzed. The Kolmogorov–Smirnov test was used to determine the normal distribution of the data. The continuous variables were expressed as mean ± SD. The one-way analysis of variance was used to compare the mean of the normally distributed parameters; otherwise, the nonparametric Kruskal–Wallis H-test was utilized. The categorical parameters were expressed as percentages and absolute frequencies. Chi-square test was utilized to compare this type of data. Spearman Rank correlation analysis was performed. A Fifty-six patients with growth in CSF culture were included in this study. 55.4% of the cases were female and 44.6% male. The youngest of the cases was 1, and the oldest was 216 months old. The mean age was 24.32 (± 55.79). Ventriculoperitoneal shunt was detected in 82.1% of the cases, and EVD catheter-related infection was found in 17.9%. Gram-positive bacteria were grown in 67.9% of the cases and gram-negative bacteria in 32.1%. When the infection and CSF biochemistry parameters of gram-positive and gram-negative bacteria were compared, no significant difference was observed .Klebsiella pneumoniae and Enterococci; 7.1% of S. aureus; 5.4% of Acinetobacter baumannii, Pseudomonas aeruginosa, and Serratia marcescens; and 1.8% of Bacillus spp., Brevundimonas diminuta, Escherichia diminutoantea, Kocrychia cocuricaelus, Koccurichia cocurica, Streptococcus salivarius, and Granuli elegans.The prevalence rates of the bacteria are as follows: 41.1% of CoNS; 10.7% of p = 0.046) and SII was significantly higher in the non-CoNS gram-positive bacteria (p = 0.002). Also, NLR and PLR were significantly higher in the non-CoNS gram-positive bacteria (p = 0.007 and p = 0.008)  .p = 0.001, p = 0.006, p = 0.044, respectively). Gram-negative bacteria have a high resistance to ampicillin, 3rd and 4th generation cephalosporin, broad-spectrum beta-lactamase   .p = 0.571). Antibiotic revision was required in 16.1% of the patients. Revision was required once in 7.1%, twice in 7.1%, and thrice in 1.8%. Only one patient (1.8%) required intrathecal treatment. The mortality rate was 19.6%. Excluding the patients who did not respond to treatment , the mean duration of treatment was 20.42 ± 11.45 days. The mean duration of treatment was 21.69 ± 9.44 days in the gram-negative bacteria group, 23.77 ± 12.57 days in the gram-positive and non-CoNS group, and 17.26 ± 11.67 days in the gram-positive and coagulase-negative group, indicating no statistically significant difference (p = 0.263). No statistical difference was found in all groups regarding the need for multiple antibiotics, the need for antibiotic revision, mortality, and response time to treatment.Single antibiotics were started in 32.1% of the patients, double antibiotics in 66.1%, and triple antibiotics in 1.8% , CSF glucose 28.36 ± 21.79 mg/dL (1-91), and CSF protein 3887.96 ± 5296.28 mg/dL (4-20723). The lowest CSF glucose level and simultaneous CSF/blood glucose ratio were observed in the non-CoNS gram-positive bacteria group, whereas the highest CSF glucose level and simultaneous CSF/blood glucose ratio were observed in the CoNS group, indicating a statistically significant difference (p = 0.009–0.046.)Catheter-related central nervous system infections, especially V-P shunt infections, are more common in the pediatric age group . CSF bioS. aureus 25% of the bacterial agents . However, in the non-CoNS gram-positive bacteria group, an inverse correlation was found between mortality and SII, which was not statistically significant. However, it was observed that the increased SII was significantly associated with mortality in the CoNS and gram-negative bacteria groups (p < 0.05). Gram-negative bacteria and CoNS usually cause benign and slow infections. The fact that the SII was higher in gram-negative bacteria and CoNS patients with mortality than in survivors may be due to the more severe and progressive gram-negative infections in the mortality group. Moreover, the reason SII was not different between survivors and patients with mortality in the non-CoNS gram-positive bacteria group and the SII was higher compared with the other groups may be due to the fact that these infections were more aggressive and rapid.CoNS is a group of bacteria found in the skin mucosa, which is considered apathogenic in healthy individuals. However, it is a common cause of hospital infections and catheter-related infections. It usually presents as subacute and chronic infections that start subclinically. However, it can have an aggressive course and be fatal in those who are inadequately treated. It should be considered in the foreground of foreign body-related infections . It was S. aureus, broad-spectrum beta-lactamase resistance and carbapenem resistance cause problems in gram-negative bacteria (Antibiotic resistance changes the treatment response and modalities. While methicillin resistance is prominent in both CoNS and bacteria . In the bacteria , it was The limitation of the our study is the small number of samples analyzed.Early and appropriate antibiotic therapy reduces morbidity and mortality in catheter-related infections. However, it is essential to start empirical antibiotic therapy until the culture results are determined. Therefore, further research on the estimation of possible factors is important. In the present study, it was determined that the level of SII upon admission was mostly parallel with non-CoNS gram-positive bacteria. However, high SII upon presentation was associated with poor prognosis and increased mortality in both the CoNS and gram-negative bacteria groups. Furthermore, no bacterial difference was observed in the shunt or EVD-related infections.10.7717/peerj.15905/supp-1Data S1Click here for additional data file.