The KPs (kisspeptins) are a family of multifunctional peptides with established roles in cancer metastasis, puberty and vasoconstriction. The effects of KPs on endothelial cells have yet to be determined. The aim of the present study was to investigate the effects of KP-10 on endothelial cell growth and the mechanisms underlying those effects. The administration of recombinant KP-10 into the hindlimbs of rats with ischaemia significantly impaired blood flow recovery, as shown by laser Doppler, and capillary growth, as shown using histology, compared with the controls. HUVECs (human umbilical vein endothelial cells) express the KP receptor and were treated with KP-10 in culture studies. KP-10 inhibited endothelial cell tube formation and proliferation in a significant and dose-dependent manner. The HUVECs treated with KP exhibited the senescent phenotype, as determined using a senescence-associated β-galactosidase assay, cell morphology analysis, and decreased Sirt1 (sirtuin 1) expression and increased p53 expression shown by Western blot analysis. Intriguingly, a pharmacological Rho kinase inhibitor, Y-27632, was found to increase the proliferation of HUVECs and to reduce the number of senescent phenotype cells affected by KP-10. In conclusion, KP-10 suppressed endothelial cells growth both in vivo and in vitro in the present study. The adverse effect of KP on endothelial cells was attributable, at least in part, to the induction of cellular senescence.
Impaired growth potential of endothelial cells causes progression of atherosclerosis and attenuation of the angiogenic response after ischaemic injury. KP has been detected in endothelial cells of an atherosclerotic plaque; however, the role of KP in endothelial cell function remains unknown.
KP-10 suppressed endothelial cell growth both in vivo and in vitro. The results of the present study demonstrated that the adverse effect of KP on endothelial cells was attributable to the induction of cellular senescence. This KP-induced senescence was reversed by ROCK inhibition.
KP impairs endothelial cell function. Inhibition of KP/GPR54 signalling may be a new therapeutic strategy against atherosclerosis and/or pathological angiogenic responses in ischaemic vascular diseases.
KPs (kisspeptins) are a family of peptides encoded by the KISS1 (KiSS-1 metastasis-suppressor) gene. The primary translation product of KISS1 is a 145-amino-acid polypeptide KP-145 . Proteolytic processing of this polypeptide results in the production of shorter peptides, namely KP-54, KP-14, KP-13 and KP-10 [2,3]. KP-54, also known as metastin, inhibits the metastasis of melanoma cells , and the same effect has been replicated in breast cancer cells treated with KP-10 . This ability of the KPs to inhibit cell migration was corroborated by an experiment showing a halt in the movement of primary trophoblasts, cells crucial for placental development during pregnancy, in response to KP-10 treatment. KP-10, the shortest of the KPs, is highly conserved between mice and humans, with only one conserved amino acid replacement . Another important role for KPs is their control of reproductive function. KP-10 is the most potent activator of gonadotropic hormone secretion and plays a critical role in both regulating GnRH (gonadotropin-releasing hormone) pulse generator frequency and controlling the hypothalamic–pituitary–gonadal axis .
The endothelium is a key regulator of vascular physiology, and a damaged or dysfunctional endothelium can initiate vascular atherosclerosis [6,7]. Endothelial cell dysfunction in humans is associated with several risk factors [8–10], correlates with disease progression  and portends cardiovascular events [12,13]. Endothelial cell dysfunction is associated not only with vasodilation and barrier function, but also with angiogenic properties such as the readiness of the cells to proliferate, migrate and form endothelial cell tubes .
Mead et al.  identified the mRNA transcription of KP and GPR54 [G-protein-coupled receptor 54; also known as KISS1R (KiSS-1 receptor)], the receptor for KP, in vascular cells in the human aorta, umbilical vein and coronary artery . When examining the vasoconstrictive effects in explanted blood vessels in the same study, they found the KPs to be localized to cells within the atherosclerotic plaque of the coronary artery . According to a study by Cho et al. , KP-10 inhibits the migration of HUVECs (human umbilical vein endothelial cells) by suppressing the expression of VEGF (vascular endothelial growth factor). These studies suggest that KP-10 may play a deleterious role in angiogenic processes after tissue ischaemia in atherosclerotic coronary and peripheral artery diseases. Thus KP-10/GPR54 signalling may be a new therapeutic target for treatment of ischaemic vascular diseases.
Although the studies described above support the involvement of KP-10 in endothelial cell function, the mechanisms underlying this involvement have yet to be unravelled. It also remains uncertain whether KP-10 affects the growth of endothelial cells [16,17]. To address this issue, we conducted in vitro experiments to investigate the effects of KP-10 on endothelial cell growth and explore the mechanisms underlying those effects. We also conducted in vivo experiments with the use of a femoral-artery-ligation model of rats to provide the first published data on the role of KP under ischaemic conditions.
MATERIALS AND METHODS
Rat hindlimb ischaemia model
The present study was performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. The Animal Care and Use Committee of Showa University approved the experimental protocol.
Hindlimb ischaemia was induced by ligating the right femoral arteries of 8-week-old male Wister rats under anaesthesia. The distal portion of the saphenous artery and all of the side branches were ligated along with the veins. The left hindlimb was kept intact and used as a non-ischaemic limb control. After the operation, the rats were injected with 20 μM KP-10 (n=3; Peptide Institute) or vehicle (control; n=3) intramuscularly at four sites in the ischaemic adductor muscle (0.1 ml each) daily for 1 week. Blood perfusion was assessed by LDPI (laser Doppler perfusion imaging; Omega Zone) at 2, 5 and 7 days after the operation. The blood flow distribution of the limb was mapped out as a colour-coded image directly proportional to the blood flow perfusion. After scanning, stored images were analysed to quantify the blood flow. The LDPI index was used to calculate the blood perfusion ratio of the ischaemic and non-ischaemic hindlimbs. Tissue samples were obtained from rat ischaemic adductor muscles at 7 days after surgery for immunohistochemical study.
HUVECs and adult D-HMVECs (human dermal microvascular endothelial cells) were purchased from Takara Bio. The cells were cultured at 37°C in a 5% CO2 atmosphere with supplemented endothelial growth medium (EGM-2 or EGM-2MV; Cambrex). HUVECs (passage 5) were plated at a density of 5×103 cells/cm2, cultured until 60% confluence was reached and treated with various concentrations of KP-10 (0, 0.1 and 1.0 μM) for 12 days. The medium was exchanged every 3 days to discard floating cells. The cell counts were calculated after 6 and 12 days of treatment. Similar experiments were performed in D-HMVECs (passage 3).
In a signal inhibition study conducted in parallel, similarly prepared HUVECs were treated with KP-10 (1.0 μM) for 12 days in the presence or absence of Y-27632 (1.0 μM; Wako), a pharmacological ROCK (Rho-associated kinase) inhibitor.
Immunohistochemistry was performed as described previously . Tissue sections were incubated with a specific primary antibody against CD31 at a 1:100 dilution (Dako) overnight at 4°C, washed three times in PBS and incubated with HRP (horseradish peroxidase)-labelled anti-rabbit or anti-mouse antibody (Histofine Simplestain Max PO; Nichirei). Finally, the binding antibody was visualized by 3,3′-diaminobenzidine staining followed by counter-staining with haematoxylin. Three fields from each tissue section (n=6) were randomly selected and the number of CD31-positive cells was counted in each field. To avoid over- or under-estimation of the capillaries as a consequence of myocyte atrophy or interstitial oedema, the capillary number adjusted per muscle fibre was used to compare differences in the capillary density.
Immunocytochemistry analysis of the endothelial cells was performed using a primary antibody against KP (Abcam) at a 1:100 dilution. For immunofluorescence staining, the cells were incubated with an anti-GPR54 antibody (Santa Cruz Biotechnology) at a 1:100 dilution and then incubated with an Alexa Fluor™ 488-conjugated goat anti-(rabbit IgG) secondary antibody (Life Technologies) at a 1:800 dilution.
RT–PCR (reverse transcription–PCR)
Upon reaching 70% confluence, HUVECs (passage 5) were cultured under a mimicked ischaemic condition [hypoxia (1% oxygen) and serum-free α-MEM (minimal essential medium); Invitrogen] for 6 h. Cells cultured under normoxia for 6 h in the growth medium (supplemented EGM-2) were used as controls.
Total RNA was extracted from the HUVECs using an RNA extraction kit (Takara Bio). RT–PCR for the KISS1, the KP-encoding gene, was performed as described previously . The following primers were used for human KISS1 and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) amplification: human KISS1, 5′-GCCATTAGAAAAGGTGGCCTC-3′ (sense) and 5′-TTGTAGTTCGGCAGGTCCTTC-3′ (antisense); and GAPDH, 5′-CACCACCATGGAGAAGGC-3′ (sense) and 5′-CCATCCACAGTCTTCTGA-3′ (antisense). The final PCR products were subjected to electrophoresis, and semi-quantitative RT–PCR analysis was performed using the ChemiDoc™ XRS+ system with Image Lab Software (Bio-Rad Laboratories). GAPDH levels were used for standardization. The relative gene expression levels were calculated as ratios of the measured PCR product densities (KISS1/GAPDH) in HUVECs cultured under the mimicked ischaemic condition relative to the PCR product densities in the controls.
In vitro angiogenesis assay
Tube formation experiments were conducted using an angiogenesis kit (Kurabo) as described previously . Briefly, HUVECs and human fibroblasts were mixed and seeded into 24-well plates. The cells were cultured at various concentrations of KP-10 (0, 0.1 and 1.0 μM) diluted in culture medium (n=4 in each group). After 11 days of culture, the HUVECs were fixed with 70% ethanol at 4°C and immunostained with an anti-(human CD31) antibody using BCIP (5-bromo-4-chloroindol-3-yl phosphate)/NBT (Nitro Blue Tetrazolium) (ThermoScientific) as a substrate for the secondary antibody. Digital photographs were taken for five selected fields per well under a microscope (Olympus). The vessel area was defined as the area of CD31-positive cells/total area, as estimated by an angiogenesis image analyser (Kurabo).
TUNEL and senescence-associated β-galactosidase staining
Apoptosis was assessed using a TUNEL (terminal deoxy-nucleotidyltransferase-mediated dUTP nick-end labelling) assay (Takara Bio). This method labels the free 3′-OH termini with FITC-labelled dUTP as an intermediate in fluorescence microscopy. The proportion of green fluorescent apoptotic cells within the total cell population was calculated for at least three random fields.
To detect cellular senescence, an SA-β-gal (senescence-associated β-galactosidase) assay (EMD Millipore Bioscience) was performed according to the manufacturer's instructions. Senescent cells were stained blue. The proportion of SA-β-gal-positive cells within the total cell population was calculated for at least three random fields.
Western blot analysis
Cell lysates (40 μg of protein) were subjected to SDS/PAGE (4–12% gradient gel). Proteins were transferred on to an Immuno-Blot™ PVDF membrane (0.2 μm; Life Technologies). After blocking, the membrane was incubated with primary antibodies against Sirt1 (sirtuin 1) at a 1:1000 dilution (Cell Signaling Technology), p53 at a 1:1000 dilution (Santa Cruz Biotechnology) and β-actin at a 1:5000 dilution (Abcam). The membranes were subsequently incubated with secondary antibodies at a 1:2000 dilution (Santa Cruz Biotechnology). After washing, the membranes were developed using an ECL reagent (Santa Cruz Biotechnology). Densitometric analysis was performed using ChemiDoc™ XRS+ with Image Lab Software as described above.
All data are expressed as means±S.E.M. Comparisons of parameters among three groups were performed by one-way ANOVA followed by Scheffe's multiple comparison test. Student's t test was used to compare the differences between two groups. P<0.05 was considered significant.
Impaired collateral growth in rat ischaemic muscle treated with KP
KP-10 was administered into the ischaemic limbs of rats to investigate the effect of KP-10 in tissues under ischaemic conditions. Blood perfusion recovery documented by LDPI was significantly attenuated in the KP-treated rats compared with the controls (Figure 1A; n=3 in each group). The immunohistochemical study with the anti-CD31 antibody showed that the capillary/muscle fibre ratio, an index of capillary density, was significantly lower in the KP group than in the controls at 7 days after the operation (Figure 1B; P<0.05). Hence KP impaired neovascularization after the ischaemic event in the muscle tissue in this animal study.
Impaired collateral growth in rat ischaemic muscle treated with KP
KP and GPR54 in HUVECs
We performed an in vitro study with HUVECs to further investigate the role of KP in endothelial cell function. Both KP and its receptor GPR54 were detected in HUVECs (Figure 2A). Semi-quantitative RT–PCR analysis for KISS1, the KP-encoding gene, was performed in HUVECs cultured either under normal growth conditions or mimicked ischaemic conditions (hypoxia and nutrient deprivation). The levels of KISS1 mRNA transcripts were significantly enhanced in the HUVECs cultured under the mimicked ischaemic conditions compared with those cultured under the normal conditions (Figure 2B; P<0.05). Severe ischaemia may enhance KP production as well as pro-angiogenic factors in the cells. The previous [15,20] and present studies suggest that endothelial cells may serve as an alternative source of KP and, thereby, act in a local paracrine/autocrine fashion.
KP and GPR54 in HUVECs
Attenuation of proliferation and tube formation in HUVECs treated with KP
HUVECs were immunostained with an anti-CD31 antibody in an angiogenesis assay and the coloured areas were designated as capillary growth. KP-10 treatment was found to prominently attenuate capillary network formation (Figure 3) compared with the controls (P<0.05).
Endothelial cell tube formation assay
In a previous study from another group, KP-10 exerted no effect on endothelial cell proliferation for 72 h after treatment . For further insight, we examined whether KP treatment influenced the propagation of HUVECs during long-term culture. HUVECs were cultured with KP-10 at 0.1 and 1.0 μM for 12 days (Figure 4). KP-10 gradually suppressed HUVEC proliferation in a dose-dependent fashion, exerting a significantly stronger suppressive effect compared with vehicle alone by the later stages of the culture (0.1 μM, P<0.05 and 1.0 μM, P<0.01 at day 12). KP-10 negatively regulated both endothelial cell migration and growth.
Endothelial cell proliferation assay
Endothelial cellular senescence by KP
We investigated the mechanisms underlying KP-induced endothelial cell dysfunction. TUNEL staining was performed to examine whether KP induced apoptosis of endothelial cells. The present study found no differences in the numbers of apoptotic cells among the samples treated with different concentrations of KP-10 (Supplementary Figure S1 at http://www.clinsci.org/cs/127/cs1270047add.htm). Next, we performed an SA-β-gal assay to detect senescent endothelial cells (enlarged, flattened and stained blue). The KP treatment significantly increased SA-β-gal-positive HUVECs in a dose-dependent manner (Figure 5). Endothelial cells with a premature senescence-like phenotype exhibited increased p53 and down-regulated Sirt1 [21,22]. We therefore evaluated the protein expressions of p53 and Sirt1 as molecular markers for cellular senescence. In the Western blot analysis, p53 was significantly increased (P<0.01) and Sirt1 was significantly decreased (P<0.05) in HUVECs treated with KP-10 compared with the controls (Figure 6). Hence KP-10 elicited premature senescence in endothelial cells and thereby suppressed the cell growth.
Western blot analysis for p53 and Sirt1 in HUVECs
Dermal microvascular endothelial cells are the principal parenchymal cells involved in wound angiogenesis . In response to tissue injury, the cells migrate into the local stroma and form new blood vessels. In the present study, GPR54 and KP were detected in the cultured D-HMVECs (Supplementary Figure S2A at http://www.clinsci.org/cs/127/cs1270047add.htm). Thus we examined whether KP-10 negatively modulated the growth of D-HMVECs as observed in HUVECs.
D-HMVECs were treated with KP-10 at 0.1 and 1.0 μM. Cell counting and SA-β-gal assay were performed after 6 days of treatment in the present study because D-HMVECs at passage 3 were found to propagate faster than HUVECs. There were significant reduced cell numbers (1.0 μM, P<0.05) and an increased proportion of senescent cells (0.1 μM, P<0.05 and 1.0 μM, P<0.05) in the D-HMVECs treated with KP-10 compared with the controls (Supplementary Figures S2B and S2C).
These results suggest that KP-10 may attenuate perfusion and wound healing in peripheral tissues after ischaemic injury via microvascular endothelial cell senescence.
Effect of ROCK inhibition on KP-induced endothelial cell senescence
A recent report showed that ROCK bound to and activated p53 . Y-27632, a pharmacological ROCK inhibitor, prolonged the life span of cultured human keratinocytes . To gain further insight, we examined whether Y-27632 modified the adverse effects of KP on endothelial cells. The addition of Y-27632 to HUVECs treated with KP-10 reversed the decrease in cell number (Figure 7) and significantly reduced the number of SA-β-gal-positive cells (P<0.01). The present results indicate that ROCK plays an important role in the signal pathway of KP for cellular senescence.
Cell proliferation and SA-β-gal assays of HUVECs treated with KP (1.0 μM) in the presence or absence of Y-27632
KP has been shown to attenuate angiogenesis in tumours and the placenta by inhibiting endothelial cell migration [16,17]. In a study by Cho et al. , KP-10 inhibited the migration and invasion of HUVECs, but not cellular proliferation over a 72-h culture, as determined using an MTS assay. In the present study, KP-10 inhibited neovascularization in ischaemic limb muscle and inhibited the proliferation of both HUVECS and D-HMVECs in long-term culture. Intriguingly, the mechanisms underlying impaired proliferative endothelial cell function by KP were, at least partly, attributable to the induction of premature cellular senescence.
Endothelial cell senescence has been proposed to contribute to endothelial cell dysfunction. Senescent endothelial cells have been found to accumulate in atherosclerotic plaques of human coronary arteries . Endothelial cell senescence apparently alters the expression levels of proteins associated with cellular architecture and cytoskeletal function [27–29]. HUVECs treated with KP-10 had increased SA-β-gal activity, took on an enlarged and flattened shape, and expressed Sirt1 and p53 at decreased and increased levels respectively as shown by Western blotting. These alterations can affect the motility of the cells, and motility changes in conjunction with a loss of replicative capacity may impair angiogenic capacity. Several lines of evidence have established the link between senescence and impaired angiogenesis. For example, it has been reported previously that the inhibition of telomerase reduces angiogenesis in tumour and therapeutic neovascularization models [30,31]. Others have demonstrated that the silencing of Sirt1 in endothelial cells abolishes the angiogenic properties of the cells .
p53 regulates cellular senescence and stress resistance as well as apoptosis in various cell types . Although the treatment with KP failed to induce apoptosis in HUVECs, it was found to induce endothelial cell senescence partly by way of p53 accumulation. Our group further explored the link between KP-10/GPR54 signalling and cellular senescence in the present study. In a study by Navenot et al. , KP triggered immediate and profound morphological modification of cells expressing GPR54. These changes were mediated through the activation of Rho and ROCK, a signalling pathway that also contributes to GPR54-mediated apoptosis. In another, more recent, report, ROCK was found to bind to and activate p53 . Y-27632 has been shown to prolong the lifespan of keratinocytes  and reverses a senescent feature of progenitor cells obtained from tendons . To investigate further, we attempted to inhibit ROCK in HUVECs treated with KP. As we expected, ROCK inhibition reversed the KP-induced senescent phenotype in endothelial cells and therefore the ROCK pathway appears to be involved in cellular senescence induced by KP-10/GPR54 signalling. It is therefore possible that ROCK inhibition may confer protective effects against endothelial cell dysfunction and diseases caused by KP. When tested as a therapeutic strategy against cardiovascular disease, ROCK inhibition has, in fact, been confirmed to confer benefits in animal models of coronary and cerebral vasospasm, arteriosclerosis/restenosis, ischaemia/reperfusion injury, hypertension, pulmonary hypertension, stroke and heart failure . Our group also found that the local administration of Y-27632 suppressed vascular remodelling after angioplasty in pigs .
In the present study, no samples from patients were examined to determine the contribution of KP-10 to diseases with severe ischaemic conditions, such as peripheral artery diseases. This is a limitation of the present study. However, we did find a higher expression of the KISS1 gene in endothelial cells under mimicked ischaemic conditions compared with the cells cultured under normal conditions. The results of our in vivo study demonstrated that the local administration of KP-10 impaired blood flow recovery in the ischaemic limbs of rats. Thus we speculate that the local production and concentration of KP-10 may be critical in the regulation of tissue angiogenesis after ischaemic injury in humans. Further studies in a clinical setting to explore the pathological role of KP-10 in ischaemic vascular diseases are necessary. As a first step, we would attempt to examine circulating KP-10 concentrations in peripheral blood from patients with vascular disease.
Taken together, the findings of the present study indicate that KP-10 confers anti-angiogenic effects in the vasculature by inducing endothelial cell senescence. The KP-induced senescence was reversed by Y-27632, a pharmacological inhibitor of ROCK. To the best of our knowledge, the present study is the first to demonstrate that KP elicits a senescent programme in mammalian cells. KP may become a therapeutic target against impaired angiogenic responses and endothelium repair in vascular diseases.
We thank Izumi Yamada and Ryuji Sato for their excellent technical assistance.
human dermal microvascular endothelial cell
G-protein-coupled receptor 54
human umbilical vein endothelial cell
laser Doppler perfusion imaging
terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling
Sayaka Usui, Yoshitaka Iso and Takuya Watanabe conceived and designed the study; Sayaka Usui, Yoshitaka Iso, Masahiro Sasai, Takuya Mizukami and Hiroyoshi Mori performed the experiments and collected the data; Sayaka Usui and Yoshitaka Iso analysed the data; Sayaka Usui, Yoshitaka Iso, Seiji Shioda and Hiroshi Suzuki interpreted results, and drafted, edited and revised the paper before submission; and all authors approved the final version of the paper.
This work was supported, in part, by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science [grant number 22790732 (to Y.I.)] and a grant for Collaborative Research from Showa University (to H.S.).