Aldehyde dehydrogenase 2 overexpression inhibits neuronal apoptosis after spinal cord ischemia / reperfusion injury

Aldehyde dehydrogenase 2 (ALDH2) is an important factor in inhibiting oxidative stress and has been shown to protect against renal ischemia/reperfusion injury. Therefore, we hypothesized that ALDH2 could reduce spinal cord ischemia/reperfusion injury. Spinal cord ischemia/reperfusion injury was induced in rats using the modified Zivin’s method of clamping the abdominal aorta. After successful model establishment, the agonist group was administered a daily consumption of 2.5% alcohol. At 7 days post-surgery, the Basso, Beattie, and Bresnahan score significantly increased in the agonist group compared with the spinal cord ischemia/reperfusion injury group. ALDH2 expression also significantly increased and the number of apoptotic cells significantly decreased in the agonist group than in the spinal cord ischemia/reperfusion injury group. Correlation analysis revealed that ALDH2 expression negatively correlated with the percentage of TUNEL-positive cells (r = −0.485, P < 0.01). In summary, increased ALDH2 expression protected the rat spinal cord against ischemia/ reperfusion injury by inhibiting apoptosis.

Introduction
Decompression of a previously severely compressed region of the spinal cord could induce spinal cord ischemia/reperfusion injury (SCII), which could lead to further irreversible postoperative complications, such as paraplegia or paraparesis (Taher et al., 2013;Zhu et al., 2013;Yang et al., 2015). SCII includes two phases: 1) immediate spinal cord injury related to acute ischemia; and 2) delayed spinal cord injury involving ischemic cellular death and reperfusion injury. Although the molecular mechanisms of ischemia/reperfu-sion injury (IRI) pathogenesis remain poorly understood, evidence suggests that oxidative stress caused by excessive production of reactive oxygen species during the IRI process is a critical factor in direct and subsequent cellular damage (Noiri et al., 2001;Walker et al., 2001;Colón and Miranda, 2016). Lipid peroxidation, which is an important source of oxidative stress, is an autocatalytic mechanism resulting in oxidative destruction of cellular membranes associated with production of toxic, reactive, aldehydic metabolites and cell death (Niki et al., 2005). Reactive aldehydes, such Spinal cord ischemia/reperfusion injury in rats Aldehyde dehydrogenase 2 Inhibiting cell apoptosis Acted by alcohol as 4-hydroxy-2-nonenal and malondialdehyde, the major end products of lipid peroxidation, are highly toxic and react with proteins to form various adducts, leading to dysfunctional proteins and subsequent cellular injury (Renner et al., 2005;Conklin et al., 2006;Marchitti et al., 2007). Previous studies of cardiac and renal IRI have suggested that preconditions with the effective activator aldehyde dehydrogenase 2 (ALDH 2 ) or physiological levels of ethanol provide protection for corresponding organs, which consistently correlates with the phosphorylation status of ALDH 2 , a key metabolic enzyme in the oxidation and detoxification of reactive aldehydes in a range of organs and cell types, to inhibit oxidative stress (Chen et al., 2008;Yuan et al., 2011). ALDH 2 is a mitochondrial enzyme and belongs to the ALDH gene family. ALDH 2 not only catalyzes oxidation of acetaldehyde to acetic acid in ethanol metabolism, but also acts as a key metabolic enzyme in the detoxification of other reactive aldehydes such as hydroxynonenal (Vasiliou et al., 2005). However, to the best of our knowledge, it is unknown whether ALDH 2 has neuroprotective effects by reducing apoptosis under conditions of SCII. The objective of our study was to investigate the early effect of ALDH 2 on apoptosis through alcohol administration in rats subjected to SCII.

Animals
Thirty adult, male, Sprague-Dawley rats, aged 8-12 weeks and weighing 250-270 g, were obtained from the Animal Laboratory of Second Military Medical University of China (license number: SCXK (Hu) 2013-0006). The study protocol was approved by the Animal Ethics Committee of Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, China. The experimental procedure followed the the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1986), and "Consensus Author Guidelines on Animal Ethics and Welfare" produced by the International Association for Veterinary Editors (IAVE).
The rats were randomly divided into sham operation group (sham group, n = 10), SCII group (I/R group, n = 10), and SCII with alcohol group (agonist group, n = 10). Rats in the sham group underwent surgery without injury. Rats in the I/R and agonist groups suffered from SCII using Zivin's method (Zivin et al., 1980). After successful model establishment, the agonist group received daily consumption of 2.5% alcohol (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China); the other groups were fed water (30 mL per day) for 7 days post-surgery.

SCII modeling and alcohol intervention
The rat model of SCII was performed employing the previously described Zivin's method (Zivin et al., 1980). All rats were prohibited from drinking during the morning of the surgery. Rats were deeply anesthetized with 3% sodium pentobarbital (1 mL/kg, intraperitoneal injection) and placed in a supine position. After making a 4-5-cm medial incision, the abdominal aorta was exposed at the level of the left renal artery. A total of 400 U heparin was injected 4 minutes before aortic occlusion, and spinal cord ischemia was induced by clamping the aorta with a bulldog clamp just below the left renal artery. After occlusion, femoral artery pulsation ceased, and blood flow was obstructed for 40 minutes. The bulldog clamp was removed, and the abdominal wall was closed. Successful rat model of spinal cord ischemia criteria: cessation of abdominal aorta and double Sham group: Normal spinal cord tissues; I/R group: serious damage; agonist group: mild damage. Arrows refer to injured cells. I/R: Ischemia/reperfusion.

Motor function assessment
Hind limb motor function assessment was performed at 7 days post-surgery using the Basso, Beattie, and Bresnahan (BBB) motor rating scale (Basso et al., 1995). This BBB scale was based on motor ability following SCII in a rat model. Briefly, the BBB scale was a 22-point scale from 0 to 21. The 0-point indicated no observable hind limb movement, and 21 points indicated consistent and coordinated gait with parallel paw placement of the limbs and consistent trunk stability. Locomotion was scored by two independent observers blind to experimental design.

Sacrifice time
All rats were sacrificed at 7 days post-surgery, and the spinal cords (L 2-5 ) were immediately removed to identify medullospinal pathologic changes, examine ALDH2 protein expression, and observe cell apoptosis, respectively, by hematoxylin-eosin staining, western blot assay, and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay.

Hematoxylin-eosin staining
The first segment of the spinal cord (L 2-5 ) was dissected and post-fixed in 4% paraformaldehyde overnight. After dehydration, the spinal cords were embedded in paraffin and 5-µm-thick serial coronal sections were collected. To obverse histopathological changes, the sections were further subjected to hematoxylin-eosin staining. Sections (5-μm-thick) were stained with hematoxylin solution for 5 minutes followed by 5 dips in 1% acid ethanol (1% HCl in 70% ethanol) and then rinsed in distilled water. Afterwards, the sections were stained with eosin solution for 3 minutes, followed by dehydration in an alcohol gradient and cleared in xylene. An independent observer, blinded to the experimental design, collected images using an inverted fluorescence microscope (Nikon Eclipse T3-S, Tokyo, Japan).

Western blot assay
The second of spinal cord (L 2-5 ) sample was centrifuged at 1,500 r/min for 30 minutes, after which the supernatant was collected. Determination of protein concentration was performed using the bicinchoninic acid protein assay kit. After transferring 0,15, 30, 60, 120, 180, 240, and 300 µL of bovine serum albumin (500 μg/mL) into each 2-mL centrifuge tube, 1× PBS was added to each tube to reach a volume of 300 µL. The final concentrations of bovine serum albumin in each centrifuge tube were 0, 25, 50, 100, 200, 300, 400, and 500 μg/mL. Working reagent was prepared by mixing 50 parts bicinchoninic acid reagent A with 1 part bicinchoninic acid reagent B (50:1, reagent A:B). The protein extract (20 μL) was placed in an appropriately labeled test tube, and diluted with PBS 30 times. An additional 200 μL of working reagent was added to each tube and mixed well. The tubes were covered and incubated at 37°C for 30 minutes. The spectrophotometer measured at 550 nm and was set to zero using a cuvette filled only with 1 × PBS. Sample absorbance was then measured within 10 minutes. The sample was electrophoresed in a 5% stacking gel with 80 V constant voltage for 20 minutes, followed by 110 V constant voltage for 3.5 hours. A total of 20 µL was resolved on an electrophoresis gel and then transferred onto polyvinylidene difluoride membranes. Primary antibody specific for ALDH 2 (goat monoclonal antibody) (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was diluted in Tris-buffered saline and Tween 20 (TBST), added to the membrane, and incubated for 60 minutes at room temperature. The membrane was then washed in TBST and incubated in secondary antibody (rabbit anti-goat horseradish peroxidase-labeled polyclonal antibody, 1:10,000; Santa Cruz Biotechnology) for 2 hours at room temperature, followed by TBST washing (3 times) and TBS washing (10 minutes). Protein bands were visualized using enhanced chemiluminescence reagent (Beyotime, Haimen, China), followed by exposure and were photographed to save the experimental results. The ratio of optical density values between ALDH 2 and Protein expression of ALDH 2 in the agonist group significantly increased compared with the I/R group. *P < 0.05, vs. sham group, #P < 0.05, vs. I/R group (mean ± SD, n = 10, one-way analysis of variance and Student-Newman-Keuls test). ALDH 2 : Aldehyde dehydrogenase 2; I/R: ischemia/ reperfusion; GAPDH: glyceraldehyde 3-phosphate dehydrogenase. Sham I/R Agonist BBB scores *# * GAPDH bands, which was an indicator of ALDH 2 protein expression, was measured using the Gel-Pro analyzer 4.5 (Media Cybernetics, Sarasota, FL, USA).

TUNEL assay
The third spinal cord (L 2-5 ) segment was subjected to antigen retrieval proteinase K (Roche) for 30 minutes, and then washed three times with phosphate-buffered saline (PBS). TUNEL solution preparation and staining were performed according to the assay kit instruction manual (Roche, Basel, Switzerland). Cell quantification was obtained by two observers using an inverted fluorescence microscope at 400× magnification. Cell apoptosis (%) was equal to the number of positive cells/(positive cells + negative cells) × 100%.

Statistical analysis
Statistical analysis was performed using SPSS 19.0 software (IBM, Armonk, IL, USA) and all data are expressed as the mean ± SD. Intergroup differences were analyzed by oneway analysis of variance and Student-Newman-Keuls test. Correlation analysis was examined by Pearson correlation coefficient. Significance was set to P < 0.05.

ALDH 2 overexpression affected behavior in SCII rats
Rats in the sham group did not show obvious symptoms of neurological impairments. BBB scores in the I/R (7.20 ± 0.40) and agonist groups (14.10 ± 0.80) significantly decreased compared with the sham group (20.00 ± 0.50) (P < 0.05), and BBB scores significantly improved in the agonist group compared with the I/R group (P < 0.05; Figure 1).

ALDH 2 overexpression affected histological changes in SCII rats
Hematoxylin-eosin staining results demonstrated well-maintained spinal cord tissues in the sham group, and neurons were morphologically normal with clear karyosomes, uniformly stained cytoplasm, and few vacuolar changes. In the I/R group, the number of normal neurons significantly decreased. Neurons were swollen and pyknotic, and the cytoplasm was stained dark with a great deal of vacuolization.
In the agonist group, these pathological changes were markedly reduced compared with the I/R group, and the number of normal neurons was significantly greater than in the I/R group. Most neurons were multipolar with clear karyosomes, with few vacuolar changes (Figure 2). ALDH 2 protein expression ALDH 2 protein expression was significantly improved in the agonist group (0.74 ± 0.04) compared with the I/R group (0.66 ± 0.05) (P < 0.05; Figure 3).

ALDH 2 overexpression affected apoptosis in SCII rats
TUNEL staining results demonstrated few TUNEL-positive cells in the sham group (8.47 ± 2.80). The percentage of TUNEL-positive cells was significantly higher in the I/R (31.69 ± 3.66) and agonist groups (20.45 ± 2.72) than in the sham group (P < 0.05). However, the percentage of TUNEL-positive cells was markedly decreased in the agonist group compared with the I/R group (P < 0.05; Figure 4).

Correlation between ALDH 2 expression and apoptotic rate
The Pearson correlation coefficient between ALDH 2 expression (X) and apoptotic rate (Y) was −0.485 in the three groups. Thus, ALDH 2 expression and apoptosis rate were negatively correlated (P < 0.01).

Discussion
Although adequate surgical decompression is the preferred treatment for chronic severe compression of the spinal cord and results in favorable surgical outcomes, SCII patients experience unpredictable and disastrous postoperative complications (Taher et al., 2013;Zhu et al., 2013;Yang et al., 2015). Because of the serious consequences of SCII, groups around the world have focused on how to effectively alleviate SCII. The present results demonstrated that moderate alcohol administration following SCII induction provides early protection. After establishment of a rat model of SCII, alcohol-treated rats displayed significantly preserved neurological function characterized by high BBB scores and less histological damage and apoptosis. Results further demonstrated that the protective effect was associated with ALDH 2 . ALDH 2 is a crucial enzyme involved in protecting the IRI by inhibiting oxidative stress. ALDH 2 is one of 19 members of the ALDH gene family and is localized within the mitochondria, a major site for reactive oxygen species and reactive aldehyde generation (Vasiliou et al., 2005). ALDH 2 not only catalyzes oxidation of acetaldehyde to acetic acid in ethanol metabolism, but also acts as a key metabolic enzyme involved in the detoxification of other reactive aldehydes such as hydroxynonenal (Vasiliou et al., 2005;Chen et al., 2008;Ma et al., 2011;Yuan et al., 2011;. Increasing evidence indicates that ALDH 2 plays a protective role in IRI of the heart, kidney, and brain. He et al. (2012) confirmed that the cardioprotective effects of alpha lipoic acid on IRI took place through a mechanism involving ALDH 2 activation. Ji et al. (2016) reported that ALDH 2 inhibited excessive mitophagy and increased cardiomyocyte following IRI by reducing 4-hydroxy-2-nonenal and reactive oxygen species levels. Yuan et al. (2011) and reported that ALDH 2 provides protection for kidneys against IRI by enhancing antioxidant capacity and preventing lipid peroxidation. Fu et al. (2014) believed ALDH 2 activation could reduce cerebral IRI by decreasing accumulation of reactive aldehydes concomitantly with improvements in brain injury and neurological function. Results from the study clearly showed that increased ALDH 2 expression alleviated SCII in rats, which was consistent with the above analysis in other organs. In our study, the agonist group rats, which expressed increased levels of ALDH 2 , also exhibited significantly improved neurological functions as assessed by BBB scores compared with the I/R group at 7 days post-surgery. Additionally, the pathological changes were markedly reduced, and the number of normal neurons was significantly greater in the agonist group compared with the I/R group. Most neurons were multipolar with clear karyosomes, and there were few vacuolar changes.
Although the underlying mechanisms involved in ALDH 2 protection against IRI, previous studies have shown detoxication of reactive aldehydes, reduced reactive oxygen species generation, and protection of mitochondrial function terminally inhibiting endoplasmic reticulum stress, apoptosis, and autophagy (Ma et al., 2011;Pang et al., 2015). The present study focused on the relationship between ALDH 2 and apoptosis during SCII.
Inhibition of apoptosis could reduce the extent of injury and preserve neurological function (Lee et al., 2003;, which is closely associated with reactive oxygen species accumulation during IRI (Ebert et al., 2014). The anti-apoptotic enzyme ALDH 2 has been shown to be involved in cell apoptosis induced by oxidative stress Fan et al., 2013). Ebert et al. (2014) reported that defective regulation of ALDH 2 -dependent signaling events induces cardiomyocyte apoptosis resulting from increased 4-hydroxy-2-nonenal and reactive oxygen species. When ALDH 2 expression is stimulated, levels of 4-hydroxy-2-nonenal, reactive oxygen species, and apoptosis significantly decrease (Pang et al., 2015). Other studies also confirmed that increased ALDH 2 expression induced by alcohol or the effective activator Alda-1 reduces cardiomyocyte apoptosis (Chen et al., 2008;Ge et al., 2012;Gao et al., 2015).  reported that increased ALDH 2 expression reduces renal cell apoptosis during IRI. Following liver injury, decreased ALDH 2 expression also leads to apoptosis . However, it remained to be shown whether ALDH 2 exhibits neuroprotective effects by reducing apoptosis in SCII. Results from the present study clearly showed that increased ALDH 2 expression stimulated by alcohol resulted in decreased SCII-induced apoptosis.
Although the molecular mechanisms involved in ALDH 2 and apoptosis remain poorly understood, increased ALDH 2 expression influences apoptosis, which might be associated with subsequent activation of heat shock protein 70, phosphorylation of c-Jun N-terminal kinase, and inhibition of p53 (Li et al., 2004;Harada, et al., 2005;. Further studies are needed to determine the relationship between these molecules.
Our study has several limitations that deserve special attention. First, it is necessary to improve accuracy in this experiment by increasing the number of animal models. Second, to determine long-term neurological protection by ALDH 2 through alcohol administration, future studies should increase the observation interval time after establishing the rat model of SCII. Third, this study preliminarily discussed the relationship between ALDH 2 and SCII, although further studies are needed to determine the involved mechanisms and contributions of ALDH 2 to SCII.
In summary, the neurological effects of alcohol on SCII took place through a mechanism involving, at least in part, ALDH 2 activation, which effectively inhibited apoptosis. Results from this study could provide a novel therapeutic target for treating spinal cord injury induced by ischemia/ reperfusion.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy:
Biology
Materials Science

text:
Two novel mechanisms for maintenance of stemness in mesenchymal stem cells: SCRG1/BST1 axis and cell–cell adhesion through N-cadherin☆

Summary Mesenchymal stem cells (MSCs) retain the ability to self-renew and differentiate into mesenchymal cells. Therefore, human MSCs are suitable candidates for use in regenerative medicine and cell therapies. Upon activation by tissue damage, MSCs contribute to tissue repair through a multitude of processes such as self-renewal, migration, and differentiation. However, loss of self-renewal and multi-lineage differentiation potential occurs at a high rate during cell doubling. Effective MSC therapies require the establishment of new techniques that preserve MSC multipotency after lengthy cell expansions. Here, two novel mechanisms are described for maintenance of stemness in MSCs via scrapie responsive gene 1 (SCRG1)/bone marrow stromal cell antigen-1 (BST1) ligand–receptor combination and cell–cell adhesion through N-cadherin. These two mechanisms findings provide a valuable tool for regenerative medicine and cell therapeutic methods that require the ex vivo expansion of human MSCs while maintaining native stem cell potential.

Introduction
Regenerative medicine and cell therapies have garnered attention as innovative methods for the treatment of serious diseases resulting from malfunctions or defects of the body. Cells used in these regenerative therapies include multipotent cells such as adult stem cells and embryonic stem (ES) cells. Recently, the advent of induced pluripotent stem (iPS) cells has contributed further to the development of regenerative medicine by removing ethical and immune barriers.
Mesenchymal stem cells (MSCs) are adult stem cells with the ability to differentiate into mesenchymal cells such as osteoblasts, adipocytes, chondrocytes, and fibroblasts, while retaining self-renewal and migration abilities [1]. In stem cell therapy, human MSCs are expanded in vitro and subsequently auto-implanted, eliminating the risk of immune rejection. Upon activation by tissue damage in vivo, MSCs contribute to tissue-repair through a multitude of processes such as self-renewal, migration, and differentiation. Human MSCs, first derived from bone marrow, have now been isolated from various tissues such as subcutaneous adipose, articular cartilage, and synovial membrane tissues [2][3][4][5]. Isolation of MSCs from a population of various cell types requires the use of cell surface markers. MSCs have been established that fulfill the following criteria: (i) adhering to plastic culture dishes; (ii) being positive for the markers CD73 (ecto-5 -nucleotidase, NT5E), CD90 (Thy-1 cell surface antigen, THY1), and CD105 (endoglin, ENG); (iii) being negative for the markers CD34, CD45 (leukocyte common antigen, LCA), HLA-DR, CD14, CD11b (integrin ␣M, ITGAM), CD79a (immunoglobulin-associated ␣), and CD19; and (iv) differentiating into osteoblasts, adipocytes, and chondrocytes in vitro [6]. In addition to these previously known cell surface markers, useful markers for isolating MSCs and maintaining multipotency have been identified. Recently, Mabuchi et al. reported that human MSCs that are positive for CD271 (low-affinity nerve growth factor receptor, LNGFR or p75 neurotrophin receptor, p75NTR) and CD90 and that are highly positive for CD106 (vascular cell adhesion molecule-1, VCAM1) retain high propensities for both of self-renewal and multipotency [7]. Thus, the combination marker LNGFR + THY1 + VCAM1 high+ (LTV) can be used to isolate potent human MSCs.
Because of their rarity in vivo, MSCs could be used after expansion for therapies in regenerative medicine [8]. Ex vivo expanded MSCs have been used for bone regeneration [9]. However, long-term in vitro culture of MSCs attenuates their ability for self-renewal and multipotency [10]. After a long period of ex vivo expansion, MSCs become large and flat and lose their ability to divide. In vitro expansion of MSCs is associated with gradual accumulation of senescent cells [11], telomere erosion [12], and changing phenotypes [13,14]. Thus, ex vivo expansion of MSCs seems to degrade multipotency; it is thus important to establish novel MSC expansion techniques that do not sacrifice multipotency even after long-term culture. For regenerative medicine and cell therapeutic purposes, this issue must be addressed. In this review, expression of the cell surface markers CD271 and CD106 and report novel trigger mechanisms for activation of the intracellular signaling pathway that regulates the maintenance of stem-like features, such as migration, self-renewal, and multipotency.

Identification of SCRG1 as a novel ligand in MSCs
The gene expression of a cysteine-rich cytokine-like peptide, scrapie responsive gene 1 (SCRG1, also known as stimulator of chondrogenesis 1), was found to decrease during osteoblastic differentiation of human bone marrowderived MSCs by using DNA microarray analysis [15]. Therefore, SCRG1 is expressed only in undifferentiated MSCs. While the function of SCRG1 is unknown, its expression was found to increase in the brains of mice infected with scrapie [16]. Recent studies by Dron et al. have reported that SCRG1 is involved in neurodegeneration and autophagy associated with transmissible spongiform encephalopathy due to scrapie infection [17,18]. SCRG1 is highly conserved in vertebrates and it consists of 98 amino acid residues, Figure 1 Proposed molecular mechanism for maintenance of stemness by the novel ligand-receptor combination SCRG1/BST1. SCRG1 is hypothesized to be secreted into the extracellular space by MSCs and to have autocrine/paracrine activity. The putative receptor for SCRG1 is a complex of BST1 and integrin ␤1 on the MSC cell surface. The SCRG1/BST1 axis promotes MSC migration and suppresses osteogenic differentiation through the FAK/PI3K/Akt signaling pathway. SCRG1/BST1 maintains the stemness of MSCs and CD271 expression.
including a 20-residue signal peptide at the N-terminus [19][20][21]. Therefore, SCRG1 protein is secreted into the extracellular space by bone marrow-derived MSCs, resulting in exhibition of autocrine/paracrine activities.

Identification of BST1 as a novel SCRG1 receptor
The SCRG1 receptor, which was predicted to be localized on the MSC membrane surface, consists of a complex composed of bone marrow stromal cell antigen-1 (BST1, CD157) and integrin ␤1 by co-immunoprecipitation analysis with a crosslinker ( Fig. 1) [15]. BST1 is an ectoenzyme with a glycosyl phosphatidylinositol (GPI) anchor and NADase/ADP-ribosyl cyclase activity in the CD38 family [22,23]. It has been found on the cell surface of stromal [24] and bone marrowderived cells [25], and it facilitates pre-B-cell growth and induces cell migration [26]. In a complex with integrin ␤1 or ␤2, BST1 has been shown to promote the phosphorylation of focal adhesion kinase (FAK) in studies using an agonistic monoclonal antibody [27][28][29]. Furthermore, it has also been reported that BST-1 regulate the adhesion and migration of leukocytes via phosphorylation of phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinases (MAPKs) [30,31]. However, the BST1 ligand has not yet been demonstrated in vivo, and SCRG1 represents the first putative ligand for this protein.

Migration induced by FAK/PI3K/Akt activation via the SCRG1/BST1 axis
Stimulation with human recombinant SCRG1 was enhance the phosphorylation of FAK in human bone marrow-derived MSCs; this effect was completely eliminated, however, by BST1 knockdown [15]. Generally, the phosphorylation of FAK via integrin ␤ is important for migration during cytoskeletal reorganization [32]. In fact, the SCRG1/BST1 axis promoted the migration of human bone marrow-derived MSCs through the activation of the FAK/PI3K/Akt signaling pathway via autocrine/paracrine activity ( Fig. 1) [15]. These phenomenon results suggest that the SCRG1/BST1 axis promotes MSC tissue-forming ability by stimulating and maintaining migratory activity.
Cell migration is closely related to stem cell homing. Stem cell therapy relies on the appropriate homing and engraftment capacity of stem cells. Chemokines such as monocyte chemotactic protein-1 (MCP-1/CCL2) and/or stromal cell-derived factor-1 (SDF-1/CXCL12), and their receptors such as CCR2 and CXCR4 promote the effective homing of MSCs. The activation of signal transduction pathways via chemokine stimulation, cell adhesion in the extracellular matrix by integrin plays an important role in migration [33]. In addition, MSCs migrate and adhere to fibroblasts via MCP-1 secreted from the fibroblasts [34]. MSC-fibroblast adhesion promotes the secretion of antiinflammatory cytokines such as interleukin (IL)-10 and transforming growth factor (TGF)-␤1. Interestingly, SCRG1 suppresses LPS-induced CC-chemokine ligand 22 (CCL22; also known as MDC) production through ERK1/2 activation in mouse macrophage-like cells [35]. Therefore, the migration ability of MSCs via SCRG1 and/or MCP-1 may be associated with immunosuppressive effects.
Expression of CD271 via the SCRG1/BST1 axis As described above, LTV-positive human MSCs are characterized by a high capacity for self-renewal and multipotency [7]. Maintenance of these capabilities is a prerequisite for proliferation of MSCs in vitro. The addition of SCRG1 led to the maintenance of CD271 expression and selfrenewal ability in a long-term primary culture of human bone marrow-derived MSCs [15]. The SCRG1/BST1 axis did not affect the expression of other important MSC markers, such as CD90 and CD106. Surprisingly, stimulation of SCRG1 preserved expression of CD271 and octamerbinding transcription factor-4 (OCT-4/POU5F1) in human bone marrow-derived MSCs, even after long-term passages. In addition, SCRG1 preserved osteogenic activity in human primary MSCs, even after long-term passages. These results suggest that SCRG1 maintains self-renewal and multipotency in MSCs, similar to the effect previously reported for the ES cell marker OCT-4 and the homeobox protein NANOG. OCT-4 and NANOG act as transcription factors by binding to the promoter region of the DNA (cytosine-5)-methyltransferase 1 (DNMT1) gene [36]. DNMT1 suppresses differentiation-inducing and cell proliferation-inhibiting factors p21 (Cip/Waf1) and p16 (INK4A), thereby promoting cell proliferation [36]. On the other hand, the Wnt signaling pathway is enhanced in CD271 + MSCs, inducing expression of OCT-4 and NANOG [37,38]. In experiments associated with long-term culture of primary human MSCs, the maintenance of OCT-4 expression was demonstrated by stimulation with SCRG1 [15]. Therefore, it appears that SCRG1 regulates the expression of not only CD271 but also OCT-4 in human bone marrow-derived MSCs. This strongly suggests that the SCRG1/BST1 axis maintains the stemness of MSCs. Nevertheless, the specific intracellular signaling pathways responsible for regulating the expressions of CD271 and OCT-4 are still unknown (Fig. 1).

Maintenance of MSC stemness by the SCRG1/BST1 axis
While SCRG1 maintains the osteogenic differentiation potential of MSCs, it suppresses the induction of osteoblastic differentiation (Fig. 1) [15]. This result indicates that it is the role of the SCRG1/BST1 axis to maintain an undifferentiated state in human bone marrow-derived MSCs by suppressing osteoblastic differentiation. Osteoblastic differentiation of MSCs involves the expression of cell proliferation inhibitory factor p21, which is regulated by the hypoxia inducible factor-1␣ (HIF-1␣)-TWIST pathway [39][40][41][42]. Proliferation and differentiation are conflicting phenomena. Thus, activation of the TWIST pathway by HIF-1␣ promotes osteoblastic differentiation and suppresses cell proliferation of MSCs [43]. As described above, SCRG1 maintains the expression of OCT-4 in primary cultured human bone marrow-derived MSCs [15]. Thus, SCRG1 will be involved in the maintenance of osteogenic differentiation potential by suppressing the expression of p21 through OCT-4. However, growth inhibitory mechanism via the SCRG1/BST1 axis in MSCs was not yet confirmed.
Ex vivo expansion of MSCs seems to degrade multipotency; it is thus important to establish novel MSC expansion techniques that do not sacrifice multipotency, even after long-term culture periods. Effective expansion of MSCs ex vivo may be achieved by utilizing recombinant SCRG1. However, SCRG1 also induces the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-jun N-terminal kinase (JNK) in MSCs [15]. As yet, the role of MAPK cascade activation by SCRG1/BST1 is still unclear. Among various possibilities, activation of MAPKs may be involved in CD271 expression and the ability to differentiate into other cell types. In order to take advantage of SCRG1 in regenerative medicine and cell therapy, further investigation onto the intracellular signaling pathway activated by SCRG1/BST1 is required.

Maintenance of stemness by cell-cell adhesion through cadherin Cell density-dependent CD106 expression
After homing of MSCs into on damaged tissue, cell-cell adhesion between MSCs is essential for MSC-dependent tissue regeneration in vivo. However, the adhesion molecules responsible for cell-cell adhesion and communication between MSCs under various culture conditions remain to be clarified. The expression of cell adhesion molecule CD106, an MSC marker, is dependent on the status of cell density of human bone marrow-derived MSCs. The expression level of CD106 was clearly up-regulated in MSCs grown under at high cell densities [44]. CD106 is primarily expressed in vascular endothelial cells and is involved in adhesion of lymphocytes onto vascular endothelium by binding to integrin ␣4␤1 (very late antigen-4, VLA-4) and integrin ␣4␤7 (lymphocyte Peyer's patch adhesion molecule, LPAM) on the cell surface of the lymphocytes [45,46]. CD106 expression is also induced by stimulation with inflammatory cytokines such as tumor necrosis factor (TNF)-␣ and IL-1␤. In particular, induction of CD106 expression in MSCs promotes homing towards wounds and damaged tissues [47,48]. Intriguingly, high cell density-dependent CD106 expression inhibited the migratory ability of MSCs [44]. In addition, integrin VLA-4 was expressed on the cell surface of MSCs. Therefore, the CD106induced inhibition of MSC migration may be dependent on cell-cell adhesion caused by the binding of CD106 to VLA-4.

NF-B activation by cell-cell adhesion through N-cadherin
The high cell density-induced expression of CD106 in human bone marrow-derived MSCs was clearly suppressed by IB kinase (IKK) inhibitors, suggesting that the cell densityinduced signal was dependent on the nuclear factor-B (NF-B) signaling pathway [44]. In addition, the expression of CD106 increases via activation of the NF-B pathway induced by cell-cell adhesion with N-cadherin in cultures with high cell densities [44,49]. The overexpression of fulllength N-cadherin in MSCs markedly increased high cell density-induced CD106 expression, whereas the overexpression of truncated N-cadherin lacking its intracellular domain suppressed high cell density-induced CD106 expression [49]. These results indicate that, in MSCs, high cell densityinducted expression of CD106 is mediated by cell-cell adhesion through N-cadherin (Fig. 2).
Cadherin is a major factor in adherens junctions (AJs) belonging to the calcium-dependent transmembrane protein family. E-cadherin is the main cadherin in the AJs of epithelial cells, while other cadherins, including N-cadherin, P-cadherin, R-cadherin, and VE-cadherin, form AJs in other cell types [50,51]. MSCs predominantly express N-cadherin, which is also observed in other cell types [52,53]. Ncadherin-mediated AJs are important in connective tissue physiology and are critical for the regulation of cell attachment and migration [54], wound healing [55], metastatic potential [56], and embryonic development [57,58], as well as the differentiation and formation of numerous specialized tissues, including fibrous connective tissues [59,60].
The phosphorylation status of proto-oncogene tyrosine-protein kinase Src in MSCs and found that phosphorylation of Src kinase was enhanced at high cell densities [49]. The phosphorylation of Src kinase in MSCs was markedly upregulated by the overexpression of full-length N-cadherin. In contrast, overexpression of a truncated version of N-cadherin lacking the intracellular domain did not affect Src kinase phosphorylation. In addition, the high cell density-induced CD106 expression was reduced by an Src kinase inhibitor [44]. These results indicate that the phosphorylation of Src kinase through the intracellular domain of N-cadherin plays an important role in the upregulation of CD106 by cell-cell adhesion (Fig. 2).

Ligand-independent PDGFR activation by cell-cell adhesion
The CD106 expression in MSCs is significantly suppressed by treatment with an inhibitor of the platelet-derived growth factor (PDGF) receptor (PDGF receptor, PDGFR) [49]. In general, PDGFR is activated by its ligand, PDGF. There are four PDGF isoforms (A-D), which form homo-or heterodimers (e.g., PDGF-AA, PDGF-AB, PDGF-BB, etc.) [61]. Of these, PDGF-BB exhibits the strongest activity [54] and has been approved by the Food and Drug Administration for the treatment of patients with bone defects in the oral and maxillofacial regions [62][63][64][65]. PDGF-BB is mainly produced by platelets and has been implicated in tissue repair [61]. There are two isoforms of PDGFR (␣ and ␤), which also form homo-or heterodimers (PDGFR␣/␣, ␣/␤, and ␤/␤) [61]. PDGFR␣ is reportedly expressed in MSCs and osteoblast progenitor cells, and PDGFR␣-positive cells exhibit a high osteoblastic differentiation capacity [66]. In MSCs, there is evidence that PDGF-BB promotes PDGF␣-positive cell migration into artificial bones without inhibiting osteoblastic differentiation [67]. In addition, TGF-␤1-induced osteoblastic differentiation of MSCs is synergistically enhanced by treatment with PDGF-BB [68]. Despite this, PDGF-BB did not affect the expression of CD106 in MSCs [49]. The intracellular signaling pathways in MSCs, Src tyrosine kinase and PDGFR␤ are ligand-independently activated by cell-cell adhesion through N-cadherin [44,49]. Furthermore, activation of PDGFR␤ induces the expression of CD106 through the NF-B pathway in MSCs [44,49]. Therefore, cell-cell adhesion induced by high cell densities enhances the expression of CD106 through N-cadherin and the ligand-independent activation of PDGFR␤ in human bone marrow-derived MSCs (Fig. 2).

Concluding remarks
In recent years, tissue engineering and regenerative medicine have been widely explored [69]. The dental pulp and periodontal ligament have attracted attention as a source of MSCs -which can differentiate into osteoblasts, cement blasts, fibroblasts, and vascular endothelial cellsindependent from the bone marrow and adipose tissues [70]. Differentiation of MSCs from periodontal tissue into odontoblasts has also been reported [71]. In periodontal tissue regeneration, repair of the alveolar bone and cementum is an important goal. Recently, we reported the establishment of MSC lines from salivary glands [72].
Cytokines and chemokines secreted from MSCs have been implicated in the immunosuppression and repair of damaged tissues [73][74][75][76]. In addition, the direction of MSC differentiation is regulated by stimulation with various growth factors, cytokines, and chemokines, as demonstrated in the differentiation of bone marrow-derived MSCs [77,78, and our unpublished data]. Interestingly, MSCs enhance anti-inflammatory effects by increasing the secretion of anti-inflammatory cytokines and simultaneously maintaining differentiation potential through cell-cell adhesion with fibroblasts.
MSCs contribute to tissue repair processes through cell proliferation, migration, and differentiation. The clinical use of adult organ-derived MSCs depends on obtaining sufficient cell populations for transplantation through ex vivo expansion. Our recent study suggested that the SCRG1/BST1 axis maintains stemness and the expression of CD271 as an MSC marker in human bone marrow-derived MSCs [15].
In addition, we have demonstrated that the expression of CD106 as an MSC marker is dependent on cell density [44,49]. In long-term culture of MSCs in vitro, the stemness of MSCs can be maintained by the addition and/or overexpression of SCRG1 while maintaining cell-cell adhesion (Fig. 3). We have additionally established fluorescently tagged immortalized MSC lines derived from different tissues of GFP-and tdTomato-transgenic mice [72,77]. These cell lines can be used for in vivo proliferation and differentiation of MSCs, as well as in vivo imaging studies to test cell therapies and regenerative medicine techniques, providing insight into diseases such as bone and immune disorders, fibrosis, and cancer progression or metastasis. These findings provide new insights into the molecular mechanisms of MSCs, as well as a novel perspective for methods of ex vivo expansion to maintain native stem cell potential for regenerative medicine and cell therapy.

Conflicts of interest
The authors declare no competing financial interests.


s2fieldsofstudy: Biology Medicine

text:
Neurokinin 3 receptor antagonism decreases gonadotropin and testosterone secretion in healthy men

Patients with mutations of neurokinin B (NKB) and its receptor show hypogonadotrophic hypogonadism, but there is little evidence for the importance of this pathway in reproductive function in normal men, or its functional hierarchy with kisspeptin.

delay, [2][3][4][5] whilst activating mutations in the kisspeptin receptor are associated with precocious puberty. 6 Whilst kisspeptin potently stimulates gonadotropin secretion [7][8][9][10][11] and increases LH pulse frequency in men, 9 the effects of exogenous NKB administration are unclear. In animal studies, both stimulatory and inhibitory actions of NKB on LH secretion have been observed [12][13][14] whilst it had no effect on reproductive hormone concentrations or LH pulsatility when infused for 8 hours to healthy men and women. 15 Administration of the NK3R antagonist ESN364 to normal men suppressed LH and testosterone but not FSH secretion. 16 However, the effect on LH lasted only few hours with recovery to baseline within 24 hours. 16 Antagonism of NK3R has been shown to decrease LH secretion and reduce the frequency of LH pulses in women with PCOS 17 and in gonadectomized animals, 18,19 indicating a role for NKB-NK3R signalling in the regulation of pulsatile GnRH and thus LH secretion.
Kisspeptin and NKB are co-expressed by some neurones within the hypothalamus, in humans and other species. These neurones can also co-express the opioid dynorphin and thus have been termed KNDy neurones. 20 The interaction and functional hierarchy between kisspeptin and neurokinin B are inferred from observations from preclinical and clinical studies. Thus in animal models, Kissr1 knockout mice were unable to show a stimulatory effect of the NK3R agonist senktide on LH secretion, 21 and in juvenile male monkeys, kisspeptin-10 stimulated LH secretion after desensitization with senktide infusion. 14 These results suggest the action of NKB is upstream of kisspeptin. This is supported by studies in patients with inactivating genetic defects in NKB (TAC3) and its NK3 receptor (TAC3R), showing slow GnRH secretion as inferred from low LH secretion, in whom kisspeptin-10 increased LH pulse frequency. 22 Women treated with an NK3R antagonist in a model of the mid-cycle LH surge showed largely preserved LH response to kisspeptin administration, although there were effects on timing and duration, and the relationship to circulating oestradiol concentrations was lost, 23 providing evidence that neurokinin B is functionally upstream of kisspeptin in the regulation of GnRH secretion in healthy women.
We, therefore, sought to determine whether NKB has a physiological role in the regulation of the male reproductive axis by the administration of the specific NK3R antagonist MLE4901, with detailed analysis of potential effects on GnRH/LH pulsatility, and to explore the hierarchy between NKB and kisspeptin in the control of this pathway.

| Study subjects
Six healthy men, aged 23-39 years were recruited to this study; all volunteers provided informed written consent and the study received Ethics Committee approval. Subjects had normal physical examination and serum LH, FSH and testosterone concentrations, and full blood count, renal function, electrolytes, liver function and electrocardiogram were within normal limits.

| Study drugs
The NK3R antagonist MLE4901 (previously known as AZD4901, kindly provided by AstraZeneca) was administered orally at 40 mg twice daily.
It has been shown to completely block neurokinin B agonist senktide induced calcium influx in human NK3R expressing Chinese hamster ovary cells with high selectivity. 24 This dose of MLE4901 reduced LH secretion and pulse frequency in normal women 23 and in women with Polycystic Ovary Syndrome. 17 Kisspeptin-10 was administered by an intravenous bolus at 0.3 μg/kg as previously described, 9 with the dose chosen as the lowest that caused maximal stimulation in LH secretion. 9

| Investigation of the effect of NK3R antagonism on gonadotropin and testosterone secretion
A schematic representation of the protocol is shown in Figure 1.
Volunteers were administered the NK3R antagonist for 7 days. Single time point daily peripheral venous blood was sampled for LH, FSH and testosterone measurement between 08.00 and 10.00 hours at 24 hours before treatment started (day −1) and on days 2, 4, 6 and 7 of NK3R antagonist administration. During treatment, the daily blood sampling was performed immediately prior to the next dose of NK3R antagonist, that is 12 hours after the previous dose.

| Investigation of the effect of NK3R antagonist on LH pulsatility
On day −1 and on the last day of NK3R antagonist administration (day 7), volunteers attended our clinical research facility for 8 hours. On F I G U R E 1 Study protocol. Six healthy men were administered the NK3R antagonist MLE4901 orally for 7 days. LH, FSH and testosterone were measured on days −1, 2, 4, 6 and 7 during the study. LH pulsatility was assessed for 6 hours on day −1 and on day 7, the last day of NK3R antagonist treatment. Kisspeptin (KP)-10 was administered by intravenous bolus at 6 hours with further frequent blood sampling for 2 hours on day −1 and day 7. [Colour figure can be viewed at wileyonlinelibrary.com] day 7, volunteers took the NK3R antagonist within an hour before starting the pulsatility assessment blood sampling. All visits commenced between 08.00 and 09.00 hours. Blood samples were collected via an indwelling intravenous cannula at 10-minute intervals for 6 hours for assessment of LH pulsatility.

| Establishment of the hierarchy between kisspeptin and neurokinin B in the regulation of LH secretion
Kisspeptin-10 was administered at 6 hours of the pulsatility study described above, with blood sampling every 10 minutes for 2 hours after administration.
Safety blood tests including full blood count, renal function, electrolytes and liver function were checked before commencing MLE4901, at the end of 7-day treatment and 2-3 weeks later. Post-treatment testosterone concentrations were measured 2 weeks after treatment.

| Hormone assays
Blood samples were centrifuged and serum frozen at −20°C or below until analysis. LH and FSH were measured by ELISA as previously described. 9 Testosterone was measured by liquid chromatographytandem mass spectrometry. LH pulsatility was assessed by deconvolution analysis on blinded data with ApEn quantified as a measure of secretory regularity. 25

| Statistical analysis
Mean LH, FSH and testosterone concentrations over time were compared using one-way analysis of variance (ANOVA) with repeated measures followed by Bonferroni's post hoc correction for multiple comparisons. Area under the curve (AUC) of LH during frequent sampling was determined by trapezoid integration on day −1 (control) and on day 7 of NK3R antagonist administration, and the change in AUC LH during one hour prekisspeptin-10 injection and for the 2 consecutive hours thereafter was calculated. Comparisons across both time and between treatments were performed using repeated measures two-way ANOVA with Bonferroni's multiple comparisons post hoc analysis. Paired Student's t test (for parametric data) or Wilcoxon matched-pairs signed rank test (for nonparametric data) were used to assess changes in measures of LH pulsatility. Data are presented as mean ± SEM.

| Ethical approval
The study protocol was approved by South East Scotland Research Ethics Committee (Ref: 09/S1101/67).

| NK3R antagonist decreases gonadotropin and testosterone secretion
LH secretion based on daily LH values decreased during NK3R antagonist administration (P < .03), with a biphasic response: LH concentrations were significantly reduced after 24 hours of treatment (4.5 ± 0.6 IU/L on day −1 to 1.7 ± 0.2 IU/L on day 2, P < .05) and then recovered (4.2 ± 0.7 IU/L on day 4 and 3.7 ± 0.7 IU/L on day 6) but were again decreased on day 7 (2.5 ± 0.6 IU/L vs day −1, P < .05) ( Figure 2A). To detect subtle changes in hormone secretion over time potentially overlooked by performing single time blood sampling, a F I G U R E 2 LH, FSH and testosterone response to administration of NK3R antagonist in healthy men (n = 6). (A) Mean of daily LH concentrations during 7 days of NK3R antagonist treatment and (B) LH AUC during 6 hours of 10-minute LH sampling on day-1 and on day 7 of NK3R antagonist administration. Mean FSH (C) and testosterone (D) response to NK3R antagonist administration. Data are presented as mean ± SEM. *P < .05; **P < .01; ***P < .001; ****P < .0001 vs day −1 more detailed analysis of LH secretion every 10 minutes for 6 hours post-NK3R antagonist dose on day 7 of treatment showed a decrease in LH AUC throughout the 6 hours post-treatment compared to day −1 (16.8 ± 2.7 vs 8.7 ± 1.5 IU/L over 6 hours with NK3Ra, P = .0007, Figure 2B).
FSH secretion was also suppressed during NK3R antagonist administration (P = .001), being significantly lower after 24 hours of treatment (3.6 ± 0.3 vs 2.4 ± 0.4 IU/L, P < .001) ( Figure 2C). FSH showed a comparable pattern to changes in LH over the following days, being significantly lower than day −1 on days 2, 4 and 7 (each P < .05 vs day −1) but not on day 6 of NK3R antagonist administration.
Testosterone secretion declined rapidly in response to NK3R antagonist at 24 hours (18.4 ± 1.6 day −1 vs 5.6 ± 1.5 nmol/L, P < .01) but in contrast to LH, was consistently suppressed for the remainder of the treatment period (P < .05 on days 4, 6 and 7 vs day −1) ( Figure 2D).
Serum testosterone recovered to pretreatment concentrations in all subjects 2 weeks later (19.8 ± 2.0 nmol/L).

| Effect of NK3R antagonist on pulsatile LH secretion in men
The characteristics of the pulsatile secretion of LH were determined before and after 7 days of NK3R antagonist treatment. The LH pulse profile from all individual subjects is shown in Figure 3. LH pulse frequency was unchanged by NK3R antagonist (0.50 ± 0.09 vs 0.47 ± 0.07 pulses/hour, ns) ( Figure 4A). However, consistent with  Figure 4D) was not significantly changed (P < .09). NK3R antagonist increased the orderliness of the LH secretion pattern as assessed by approximate entropy (ApEn) (P = .02; Figure 4E).

| NK3R antagonist does not reduce kisspeptininduced LH secretion
To assess the hierarchical interaction between kisspeptin and neurokinin B in the regulation of LH secretion in men, an intravenous bolus injection of kisspeptin-10 was administered after 6 hours of blood sampling on day −1 and on day 7 of NK3R antagonist treatment ( Figure 5). Kisspeptin-10 elicited a rapid increase in LH concentration (3.0 ± 0.6 at 6 hours vs 4.8 ± 0.5 IU/L at 7 hours, P < .05 and AUC LH 2.7 ± 0.4 at 6 hours vs 5.2 ± 0.5 IU/L hour at 7 hours, P < .01;

| Tolerability and safety
MLE4901 treatment was well tolerated. One man reported reduced libido whist on NK3R antagonist and this recovered after the completion of the study. Haematology and biochemistry safety parameters remained stable in all subjects throughout the study period.

| DISCUSSION
The present study investigated the role of neurokinin B in the control of the reproductive axis in healthy men and its interaction with kisspeptin in the regulation of GnRH secretion, using LH as a surrogate.
F I G U R E 4 Analysis of 6 hour LH secretory pattern on day 7 of NK3R antagonist treatment compared to control day −1 in healthy men. Mean LH pulse frequency (A), basal (nonpulsatile) LH secretion (B), pulsatile LH secretion (C), LH secretory mass per pulse (D) and the relative orderliness/regularity of LH secretory pattern (E) on day 7 of NK3R antagonist treatment compared to day −1. Data are presented as mean ± SEM. *P = .02 Pharmacological blockade of NKB-NK3R signalling caused a rapid and marked decrease in LH, FSH and testosterone secretion. However, the stimulatory LH response to kisspeptin after 7 days of NK3R antagonist was maintained. NK3R antagonism did not affect the frequency of LH pulses in men, but other aspects of the pulsatile nature of LH secretion were markedly reduced. These data provide novel evidence that NKB is a major regulator of the physiological regulation of GnRH/ LH and testosterone secretion in men. They also support a predominantly hierarchical relationship whereby central action of neurokinin B is proximal to kisspeptin in the modulation of GnRH/LH secretion, as previously indicated in nonhuman primates, 14 in men with NKB/NK3R mutations 22 and in healthy women. 23 These data demonstrate that in men, NK3R antagonist administration decreased LH secretion in a biphasic pattern, with a marked reduction after 24 hours administration (on day 2), and on day 7. Consistent with this being by suppression of GnRH secretion, NK3R antagonist also lowered FSH concentrations on days 2, 4 and 7 of treatment, albeit to a lesser extent than LH secretion. NK3R antagonists have been reported to suppress LH secretion in ovariectomized ewes (MRK-08) and castrate monkeys (ESN364) 18 NKB-NK3R mRNA has been detected in human granulosa cells. 26,27 However, NKB-NK3R mRNA has not been reported in human testis. It is, however, plausible that the initial fall in testosterone results in reduced negative feedback with a secondary recovery in LH secretion, although caution must be taken when interpreting those findings given that once only daily blood sampling may miss subtle changes in hormone secretion especially when there are relatively rapid changes in steroid feedback and the pathways that mediate that feedback are likely to be impacted by this drug. Nevertheless, analysis over 6 hours after dosing on day 7 showed consistent suppression of LH suggesting that suppression would persist with chronic antagonism of NKB-NK3R signalling. FSH secretion is less sensitive to rapid changes in GnRH secretion, which may account for the slightly different profile of the FSH response to NK3R antagonism over the 7 days of treatment.
The present study has shown that in normal men, NK3R antagonist administration markedly reduced both basal (ie nonpulsatile) LH secretion and the total amount of LH secreted in pulses, contributing to overall lower serum LH concentrations. The regularity of the LH secretory pattern and by inference GnRH secretion, showed greater orderliness, as indicated by reduced ApEn. NK3R antagonist did not alter the frequency of LH pulses, although the ability to detect a change in LH pulse frequency may have been limited by the small number of men studied here and the limited duration of frequent sampling. This contrasts with data from administration of the same NK3R antagonist to women with PCOS 17 and to normal women in a model of the LH F I G U R E 5 LH response to kisspeptin-10 injection with and without NK3R antagonist pretreatment in healthy men. (A) Mean LH concentrations from 5 to 8 hours of frequent sampling following kisspeptin-10 administration at hour 6; (B) AUC LH and (C) ΔAUC LH were compared over one hour before kisspeptin-10 administration (hour 6), with 2 hours postkisspeptin-10 administration (hours 7 and 8), with (day 7) and without (day −1) NK3R antagonist pretreatment during frequent blood sampling for LH every 10 minutes. Data are presented as mean ± SEM. *P < .05; **P < .01; ****P < .0001 surge 23 and to administration of different NK3R antagonists (MRK-08 and ESN364) to ovariectomized ewes, 18,19 where reductions in LH pulse frequency were demonstrated. This may, however, reflect sexual dimorphism in that changes in the frequency of LH pulses are not seen in male reproduction, 28 unlike the changes in LH pulse frequency that characterize (but are not essential for) the normal menstrual cycle. 29,30 Sexual dimorphism is well recognized in the kisspeptin-induced LH response, with a marked increase in LH in men but a variable response in women dependent on the sex steroid milieu. 31,32 The female hypothalamus has significantly more kisspeptin cell bodies and fibres compared to men, 33 and there is a greater abundance of neurokinin B-expressing neurones in ewes than rams, 34  in response to kisspeptin-10 in the presence of the NK3R antagonist, but this is likely to reflect the lower basal LH concentrations following 7 days of NK3R antagonist administration. There was no difference in mean LH levels after kisspeptin-10 administration alone or following NK3R antagonist treatment. NKB may modulate kisspeptin action by altering the sensitivity of GnRH neurones to the latter and/or by acting on GnRH terminals enhancing, either synergistically or additively, kisspeptin effects. Although NKB/Kp immunoreactive terminals are in close apposition to GnRH terminals in the median eminence, 36 no NK3R expression on GnRH neurones has been showed in mice and ewes. 37,38 Direct action of NKB on GnRH neurones therefore seems unlikely.
Conversely, co-infusion of neurokinin B itself and kisspeptin-54 in healthy men stimulated gonadotropin and testosterone secretion significantly less than with kisspeptin-54 alone and had no effect on LH pulsatility. 39 As reductions in GnRH and Kiss1r mRNA were observed after administration of the NKB agonist senktide in rats, 40 it was proposed that NKB can act to inhibit GnRH and kisspeptin transcription to reduce the stimulatory action of kisspeptin on gonadotrophins. 39 Gonadotropin inhibitory hormone (GnIH) also appears to be upstream of kisspeptin, having no suppressive effect on kisspeptin-induced LH secretion in a recent study in men, 41 although its interaction with neurokinin B on modulating GnRH secretion remains unknown. Whilst it is possible that NKB exerts variable stimulatory and/or inhibitory downstream actions on kisspeptin signalling, perhaps in response to the sex steroid feedback environment, the present data clearly support a stimulatory role of NKB signalling in the normal male HPG axis.
The present study has strengths in that a specific neurokinin-3 receptor antagonist was used, detailed LH pulse profiling was performed with blinded pulse analysis, and paired data were obtained for the control period and during administration of NK3R antagonist. The lack of suppression in the frequency of LH pulses by the NK3R antagonist in men is in contrast to the same dose of MLE4901 reducing LH pulse frequency in women with PCOS 17 and in normal women, 23 which may reflect sexual dimorphism. However, the sample size is small, and a placebo control was not employed in this mechanistic exploratory study.
In summary, we have shown that NK3R antagonism resulted in suppression of both LH and FSH concentrations, and a sustained decrease in testosterone secretion in healthy men over a 7-day period, indicating an important role for neurokinin B in the physiological regulation of the male HPG axis. We showed that the NK3R antagonist reduced some aspects of pulsatile LH secretion in men. Assessment of the interaction between neurokinin B and kisspeptin showed that LH response to kisspeptin was maintained in the presence of NK3R antagonist, supporting a functionally hierarchical relationship of neurokinin B being predominantly proximal to kisspeptin. These data thus indicate that neurokinin B modulates GnRH/LH secretion in men, with potential for therapeutic development as well as exploration of its contribution to disorders of reproductive function.