Short-term hypercaloric carbohydrate loading increases surgical stress resilience by inducing FGF21.

Dietary restriction promotes resistance to surgical stress in multiple organisms. Counterintuitively, current medical protocols recommend short-term carbohydrate-rich drinks (carbohydrate loading) prior to surgery, part of a multimodal perioperative care pathway designed to enhance surgical recovery. Despite widespread clinical use, preclinical and mechanistic studies on carbohydrate loading in surgical contexts are lacking. Here we demonstrate in ad libitum-fed mice that liquid carbohydrate loading for one week drives reductions in solid food intake, while nearly doubling total caloric intake. Similarly, in humans, simple carbohydrate intake is inversely correlated with dietary protein intake. Carbohydrate loading-induced protein dilution increases expression of hepatic fibroblast growth factor 21 (FGF21) independent of caloric intake, resulting in protection in two models of surgical stress: renal and hepatic ischemia-reperfusion injury. The protection is consistent across male, female, and aged mice. In vivo, amino acid add-back or genetic FGF21 deletion blocks carbohydrate loading-mediated protection from ischemia-reperfusion injury. Finally, carbohydrate loading induction of FGF21 is associated with the induction of the canonical integrated stress response (ATF3/4, NF-kB), and oxidative metabolism (PPARγ). Together, these data support carbohydrate loading drinks prior to surgery and reveal an essential role of protein dilution via FGF21.

Sodium Hydrosulfide Treatment During Porcine Kidney Ex Vivo Perfusion and Transplantation.

Background: In rodents, hydrogen sulfide (H2S) reduces ischemia-reperfusion injury and improves renal graft function after transplantation. Here, we hypothesized that the benefits of H2S are conserved in pigs, a more clinically relevant model. Methods: Adult porcine kidneys retrieved immediately or after 60 min of warm ischemia (WI) were exposed to 100 µM sodium hydrosulfide (NaHS) (1) during the hypothermic ex vivo perfusion only, (2) during WI only, and (3) during both WI and ex vivo perfusion. Kidney perfusion was evaluated with dynamic contrast-enhanced MRI. MRI spectroscopy was further employed to assess energy metabolites including ATP. Renal biopsies were collected at various time points for histopathological analysis. Results: Perfusion for 4 h pig kidneys with Belzer MPS UW + NaHS resulted in similar renal perfusion and ATP levels than perfusion with UW alone. Similarly, no difference was observed when NaHS was administered in the renal artery before ischemia. After autotransplantation, no improvement in histologic lesions or cortical/medullary kidney perfusion was observed upon H2S administration. In addition, AMP and ATP levels were identical in both groups. Conclusions: In conclusion, treatment of porcine kidney grafts using NaHS did not result in a significant reduction of ischemia-reperfusion injury or improvement of kidney metabolism. Future studies will need to define the benefits of H2S in human, possibly using other molecules as H2S donors.

Cystathionine Gamma Lyase Is Regulated by Flow and Controls Smooth Muscle Migration in Human Saphenous Vein.

The saphenous vein is the conduit of choice for bypass grafting. Unfortunately, the hemodynamic stress associated with the arterial environment of the bypass vein graft leads to the development of intimal hyperplasia (IH), an excessive cellular growth and collagen deposition that results in restenosis and secondary graft occlusion. Hydrogen sulfide (H2S) is a ubiquitous redox-modifying gasotransmitter that inhibits IH. H2S is produced via the reverse trans-sulfuration pathway by three enzymes: cystathionine γ-lyase (CSE), cystathionine ß-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST). However, the expression and regulation of these enzymes in the human vasculature remains unclear. Here, we investigated the expression of CSE, CBS and 3-MST in segments of native human saphenous vein and large arteries. Furthermore, we evaluated the regulation of these enzymes in vein segments cultured under static, venous (7 mmHg pressure) or arterial (100 mmHg pressure) pressure. CSE was expressed in the media, neointima and intima of the vessels and was negatively regulated by arterial shear stress. Adenoviral-mediated CSE overexpression or RNA interference-mediated CSE knock-down revealed that CSE inhibited primary human VSMC migration but not proliferation. We propose that high shear stress in arteriovenous bypass grafts inhibits CSE expression in both the media and endothelium, which may contribute to increased VSMC migration in the context of IH.

Clinical Potential of Hydrogen Sulfide in Peripheral Arterial Disease.

Peripheral artery disease (PAD) affects more than 230 million people worldwide. PAD patients suffer from reduced quality of life and are at increased risk of vascular complications and all-cause mortality. Despite its prevalence, impact on quality of life and poor long-term clinical outcomes, PAD remains underdiagnosed and undertreated compared to myocardial infarction and stroke. PAD is due to a combination of macrovascular atherosclerosis and calcification, combined with microvascular rarefaction, leading to chronic peripheral ischemia. Novel therapies are needed to address the increasing incidence of PAD and its difficult long-term pharmacological and surgical management. The cysteine-derived gasotransmitter hydrogen sulfide (H2S) has interesting vasorelaxant, cytoprotective, antioxidant and anti-inflammatory properties. In this review, we describe the current understanding of PAD pathophysiology and the remarkable benefits of H2S against atherosclerosis, inflammation, vascular calcification, and other vasculo-protective effects.

Cystathionine-γ-lyase overexpression modulates oxidized nicotinamide adenine dinucleotide biosynthesis and enhances neovascularization.

Objective: Hydrogen sulfide is a proangiogenic gas produced primarily by the transsulfuration enzyme cystathionine-γ-lyase (CGL). CGL-dependent hydrogen sulfide production is required for neovascularization in models of peripheral arterial disease. However, the benefits of increasing endogenous CGL and its mechanism of action have not yet been elucidated. Methods: Male whole body CGL-overexpressing transgenic (CGLTg) mice and wild-type (WT) littermates (C57BL/6J) were subjected to the hindlimb ischemia model (age, 10-12 weeks). Functional recovery was assessed via the treadmill exercise endurance test. Leg perfusion was measured by laser Doppler imaging and vascular endothelial-cadherin immunostaining. To examine the angiogenic potential, aortic ring sprouting assay and postnatal mouse retinal vasculature development studies were performed. Finally, comparative metabolomics analysis, oxidized/reduced nicotinamide adenine dinucleotide (NAD+/NADH) analysis, and quantitative real-time polymerase chain reaction were performed on CGLWT and CGLTg gastrocnemius muscle. Results: The restoration of blood flow occurred more rapidly in CGLTg mice. Compared with the CGLWT mice, the median ± standard deviation running distance and time were increased for the CGLTg mice after femoral artery ligation (159 ± 53 m vs 291 ± 74 m [P < .005] and 17 ± 4 minutes vs 27 ± 5 minutes [P < .05], respectively). Consistently, in the CGLTg ischemic gastrocnemius muscle, the capillary density was increased fourfold (0.05 ± 0.02 vs 0.20 ± 0.12; P < .005). Ex vivo, the endothelial cell (EC) sprouting length was increased in aorta isolated from CGLTg mice, especially when cultured in VEGFA (vascular endothelial growth factor A)-only media (63 ± 2 pixels vs 146 ± 52 pixels; P < .05). Metabolomics analysis demonstrated a higher level of niacinamide, a precursor of NAD+/NADH in the muscle of CGLTg mice (61.4 × 106 ± 5.9 × 106 vs 72.4 ± 7.7 × 106 area under the curve; P < .05). Similarly, the NAD+ salvage pathway gene expression was increased in CGLTg gastrocnemius muscle. Finally, CGL overexpression or supplementation with the NAD+ precursor nicotinamide mononucleotide improved EC migration in vitro (wound closure: control, 35% ± 9%; CGL, 55% ± 11%; nicotinamide mononucleotide, 42% ± 13%; P < .05). Conclusions: Our results have demonstrated that CGL overexpression improves the neovascularization of skeletal muscle on hindlimb ischemia. These effects correlated with changes in the NAD pathway, which improved EC migration.

Sodium thiosulfate, a source of hydrogen sulfide, stimulates endothelial cell proliferation and neovascularization.

Therapies to accelerate vascular repair are currently lacking. Pre-clinical studies suggest that hydrogen sulfide (H2S), an endogenous gasotransmitter, promotes angiogenesis. Here, we hypothesized that sodium thiosulfate (STS), a clinically relevant source of H2S, would stimulate angiogenesis and vascular repair. STS stimulated neovascularization in WT and LDLR receptor knockout mice following hindlimb ischemia as evidenced by increased leg perfusion assessed by laser Doppler imaging, and capillary density in the gastrocnemius muscle. STS also promoted VEGF-dependent angiogenesis in matrigel plugs in vivo and in the chorioallantoic membrane of chick embryos. In vitro, STS and NaHS stimulated human umbilical vein endothelial cell (HUVEC) migration and proliferation. Seahorse experiments further revealed that STS inhibited mitochondrial respiration and promoted glycolysis in HUVEC. The effect of STS on migration and proliferation was glycolysis-dependent. STS probably acts through metabolic reprogramming of endothelial cells toward a more proliferative glycolytic state. These findings may hold broad clinical implications for patients suffering from vascular occlusive diseases.

Subnormothermic Ex Vivo Porcine Kidney Perfusion Improves Energy Metabolism: Analysis Using 31P Magnetic Resonance Spectroscopic Imaging.

The ideal preservation temperature for donation after circulatory death kidney grafts is unknown. We investigated whether subnormothermic (22 °C) ex vivo kidney machine perfusion could improve kidney metabolism and reduce ischemia-reperfusion injury. Methods: To mimic donation after circulatory death procurement, kidneys from 45-kg pigs underwent 60 min of warm ischemia. Kidneys were then perfused ex vivo for 4 h with Belzer machine perfusion solution UW at 22 °C or at 4 °C before transplantation. Magnetic resonance spectroscopic imaging coupled with LCModel fitting was used to assess energy metabolites. Kidney perfusion was evaluated with dynamic-contrast enhanced MRI. Renal biopsies were collected at various time points for histopathologic analysis. Results: Total adenosine triphosphate content was 4 times higher during ex vivo perfusion at 22 °C than at 4 °C perfusion. At 22 °C, adenosine triphosphate levels increased during the first hours of perfusion but declined afterward. Similarly, phosphomonoesters, containing adenosine monophosphate, were increased at 22 °C and then slowly consumed over time. Compared with 4 °C, ex vivo perfusion at 22 °C improved cortical and medullary perfusion. Finally, kidney perfusion at 22 °C reduced histological lesions after transplantation (injury score: 22 °C: 10.5 ± 3.5; 4 °C: 18 ± 2.25 over 30). Conclusions: Ex vivo kidney perfusion at 22°C improved graft metabolism and protected from ischemia-reperfusion injuries upon transplantation. Future clinical studies will need to define the benefits of subnormothermic perfusion in improving kidney graft function and patient's survival.

Clinical Use of Hydrogen Sulfide to Protect Against Intimal Hyperplasia.

Arterial occlusive disease is the narrowing of the arteries via atherosclerotic plaque buildup. The major risk factors for arterial occlusive disease are age, high levels of cholesterol and triglycerides, diabetes, high blood pressure, and smoking. Arterial occlusive disease is the leading cause of death in Western countries. Patients who suffer from arterial occlusive disease develop peripheral arterial disease (PAD) when the narrowing affects limbs, stroke when the narrowing affects carotid arteries, and heart disease when the narrowing affects coronary arteries. When lifestyle interventions (exercise, diet…) fail, the only solution remains surgical endovascular and open revascularization. Unfortunately, these surgeries still suffer from high failure rates due to re-occlusive vascular wall adaptations, which is largely due to intimal hyperplasia (IH). IH develops in response to vessel injury, leading to inflammation, vascular smooth muscle cells dedifferentiation, migration, proliferation and secretion of extra-cellular matrix into the vessel's innermost layer or intima. Re-occlusive IH lesions result in costly and complex recurrent end-organ ischemia, and often lead to loss of limb, brain function, or life. Despite decades of IH research, limited therapies are currently available. Hydrogen sulfide (H2S) is an endogenous gasotransmitter derived from cysteine metabolism. Although environmental exposure to exogenous high H2S is toxic, endogenous H2S has important vasorelaxant, cytoprotective and anti-inflammatory properties. Its vasculo-protective properties have attracted a remarkable amount of attention, especially its ability to inhibit IH. This review summarizes IH pathophysiology and treatment, and provides an overview of the potential clinical role of H2S to prevent IH and restenosis.

Sodium thiosulfate acts as a hydrogen sulfide mimetic to prevent intimal hyperplasia via inhibition of tubulin polymerisation.

BACKGROUND: Intimal hyperplasia (IH) remains a major limitation in the long-term success of any type of revascularisation. IH is due to vascular smooth muscle cell (VSMC) dedifferentiation, proliferation and migration. The gasotransmitter Hydrogen Sulfide (H2S), mainly produced in blood vessels by the enzyme cystathionine- γ-lyase (CSE), inhibits IH in pre-clinical models. However, there is currently no H2S donor available to treat patients. Here we used sodium thiosulfate (STS), a clinically-approved source of sulfur, to limit IH. METHODS: Low density lipoprotein receptor deleted (LDLR-/-), WT or Cse-deleted (Cse-/-) male mice randomly treated with 4 g/L STS in the water bottle were submitted to focal carotid artery stenosis to induce IH. Human vein segments were maintained in culture for 7 days to induce IH. Further in vitro studies were conducted in primary human vascular smooth muscle cells (VSMCs). FINDINGS: STS inhibited IH in WT mice, as well as in LDLR-/- and Cse-/- mice, and in human vein segments. STS inhibited cell proliferation in the carotid artery wall and in human vein segments. STS increased polysulfides in vivo and protein persulfidation in vitro, which correlated with microtubule depolymerisation, cell cycle arrest and reduced VSMC migration and proliferation. INTERPRETATION: STS, a drug used for the treatment of cyanide poisoning and calciphylaxis, protects against IH in a mouse model of arterial restenosis and in human vein segments. STS acts as an H2S donor to limit VSMC migration and proliferation via microtubule depolymerisation. FUNDING: This work was supported by the Swiss National Science Foundation (grant FN-310030_176158 to FA and SD and PZ00P3-185927 to AL); the Novartis Foundation to FA; and the Union des Sociétés Suisses des Maladies Vasculaires to SD, and the Fondation pour la recherche en chirurgie vasculaire et thoracique.

Stapled Porcine Pericardium Displays Lower Infectivity In Vitro Than Native and Sutured Porcine Pericardium.

BACKGROUND: Biological xenografts using tubulized porcine pericardium are an alternative to replace infected prosthetic graft. We recently reported an innovative technique using a stapled porcine pericardial bioconduit for immediate vascular reconstruction in emergency. The objective of this study is to compare the growth and adherence to grafts of bacteria and yeast incubated with stapled porcine pericardium, sutured or naked pericardium. MATERIAL AND METHODS: One square centimeter of porcine pericardial patches, with or without staples or sutures, was incubated with 105 colony forming units of Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and Candida albicans for 1, 6, and 24 h. The medium was collected to quantify planktonic microorganisms, while grafts were sonicated to quantify adherent microorganisms. Dacron and Dacron Silver were analyzed in parallel as synthetic reference prostheses. RESULTS: Stapled porcine pericardium reduced the growth and the adherence of E coli (2- to 30-fold; P < 0.0005), S aureus (11- to 1000-fold; P < 0.0006), S epidermidis (>500-fold; P < 0.0001), and C albicans (12- to 50-fold; P < 0.0001) when compared to medium alone (growth) and pericardium or Dacron (adherence). Native and sutured porcine pericardium interfered with the growth and the adherence of E coli and C albicans, and Dacron with that of S epidermidis. As expected, Dacron Silver was robustly bactericidal. CONCLUSIONS: Stapled porcine pericardium exhibited a lower susceptibility to infection by bacteria and yeasts in vitro when compared to the native and sutured porcine pericardium. Stapled porcine pericardium might be a good option for rapid vascular grafting without increasing infectivity.

Vascular smooth muscle cells in intimal hyperplasia, an update.

Arterial occlusive disease is the leading cause of death in Western countries. Core contemporary therapies for this disease include angioplasties, stents, endarterectomies and bypass surgery. However, these treatments suffer from high failure rates due to re-occlusive vascular wall adaptations and restenosis. Restenosis following vascular surgery is largely due to intimal hyperplasia. Intimal hyperplasia develops in response to vessel injury, leading to inflammation, vascular smooth muscle cells dedifferentiation, migration, proliferation and secretion of extra-cellular matrix into the vessel's innermost layer or intima. In this review, we describe the current state of knowledge on the origin and mechanisms underlying the dysregulated proliferation of vascular smooth muscle cells in intimal hyperplasia, and we present the new avenues of research targeting VSMC phenotype and proliferation.

Hydrogen Sulphide Release via the Angiotensin Converting Enzyme Inhibitor Zofenopril Prevents Intimal Hyperplasia in Human Vein Segments and in a Mouse Model of Carotid Artery Stenosis.

OBJECTIVE: Hypertension is a major risk factor for intimal hyperplasia (IH) and re-stenosis following vascular and endovascular interventions. Preclinical studies suggest that hydrogen sulphide (H2S), an endogenous gasotransmitter, limits re-stenosis. While there is no clinically available pure H2S releasing compound, the sulfhydryl containing angiotensin converting enzyme inhibitor zofenopril is a source of H2S. Here, it was hypothesised that zofenopril, due to H2S release, would be superior to other non-sulfhydryl containing angiotensin converting enzyme inhibitors (ACEi) in reducing intimal hyperplasia. METHODS: Spontaneously hypertensive male Cx40 deleted mice (Cx40-/-) or wild type (WT) littermates were randomly treated with enalapril 20 mg or zofenopril 30 mg. Discarded human vein segments and primary human smooth muscle cells (SMCs) were treated with the active compound enalaprilat or zofenoprilat. IH was evaluated in mice 28 days after focal carotid artery stenosis surgery and in human vein segments cultured for seven days ex vivo. Human primary smooth muscle cell (SMC) proliferation and migration were studied in vitro. RESULTS: Compared with control animals (intima/media thickness 2.3 ± 0.33 µm), enalapril reduced IH in Cx40-/- hypertensive mice by 30% (1.7 ± 0.35 µm; p = .037), while zofenopril abrogated IH (0.4 ± 0.16 µm; p < .002 vs. control and p > .99 vs. sham operated Cx40-/- mice). In WT normotensive mice, enalapril had no effect (0.9665 ± 0.2 µm in control vs. 1.140 ± 0.27 µm; p > .99), while zofenopril also abrogated IH (0.1623 ± 0.07 µm; p < .008 vs. control and p > .99 vs. sham operated WT mice). Zofenoprilat, but not enalaprilat, also prevented IH in human vein segments ex vivo. The effect of zofenopril on carotid and SMCs correlated with reduced SMC proliferation and migration. Zofenoprilat inhibited the mitogen activated protein kinase and mammalian target of rapamycin pathways in SMCs and human vein segments. CONCLUSION: Zofenopril provides extra beneficial effects compared with non-sulfhydryl ACEi in reducing SMC proliferation and re-stenosis, even in normotensive animals. These findings may hold broad clinical implications for patients suffering from vascular occlusive diseases and hypertension.

Differential role of nicotinamide adenine dinucleotide deficiency in acute and chronic kidney disease.

BACKGROUND: Nicotinamide adenine dinucleotide (NAD+) is a ubiquitous coenzyme involved in electron transport and a co-substrate for sirtuin function. NAD+ deficiency has been demonstrated in the context of acute kidney injury (AKI). METHODS: We studied the expression of key NAD+ biosynthesis enzymes in kidney biopsies from human allograft patients and patients with chronic kidney disease (CKD) at different stages. We used ischaemia-reperfusion injury (IRI) and cisplatin injection to model AKI, urinary tract obstruction [unilateral ureteral obstruction (UUO)] and tubulointerstitial fibrosis induced by proteinuria to investigate CKD in mice. We assessed the effect of nicotinamide riboside (NR) supplementation on AKI and CKD in animal models. RESULTS: RNA sequencing analysis of human kidney allograft biopsies during the reperfusion phase showed that the NAD+de novo synthesis is impaired in the immediate post-transplantation period, whereas the salvage pathway is stimulated. This decrease in de novo NAD+ synthesis was confirmed in two mouse models of IRI where NR supplementation prevented plasma urea and creatinine elevation and tubular injury. In human biopsies from CKD patients, the NAD+de novo synthesis pathway was impaired according to CKD stage, with better preservation of the salvage pathway. Similar alterations in gene expression were observed in mice with UUO or chronic proteinuric glomerular disease. NR supplementation did not prevent CKD progression, in contrast to its efficacy in AKI. CONCLUSION: Impairment of NAD+ synthesis is a hallmark of AKI and CKD. NR supplementation is beneficial in ischaemic AKI but not in CKD models.

Targeting connexin37 alters angiogenesis and arteriovenous differentiation in the developing mouse retina.

Connexin37 (Cx37) forms intercellular channels between endothelial cells (EC), and contributes to coordinate the motor tone of vessels. We investigated the contribution of this protein during physiological angiogenesis. We show that, compared to WT littermates, mice lacking Cx37 (Cx37-/- ) featured (i) a decreased extension of the superficial vascular plexus during the first 4 days after birth; (ii) an increased vascular density at the angiogenic front at P6, due to an increase in the proliferative rate of EC and in the sprouting of the venous compartment, as well as to a somewhat displaced position of tip cells; (iii) a decreased coverage of newly formed arteries and veins by mural cells; (iv) altered ERK-dependent endothelial cells proliferation through the EphB4 signaling pathway, which is involved in the specification of veins and arteries. In vitro studies documented that, in the absence of Cx37, human venous EC (HUVEC) released less platelet-derived growth factor (PDGF) and more Angiopoietin-2, two molecules involved in the recruitment of mural cells. Treatment of mice with DAPT, an inhibitor of the Notch pathway, decreased the expression of Cx37, and partially mimicked in WT retinas, the alterations observed in Cx37-/- mice. Thus, Cx37 contributes to (i) the early angiogenesis of retina, by interacting with the Notch pathway; (ii) the growth and maturation of neo-vessels, by modulating tip, stalk, and mural cells; (iii) the regulation of arteriovenous specification, thus, representing a novel target for treatments of retina diseases.

Hydrogen sulfide-releasing peptide hydrogel limits the development of intimal hyperplasia in human vein segments.

Currently available interventions for vascular occlusive diseases suffer from high failure rates due to re-occlusive vascular wall adaptations, a process called intimal hyperplasia (IH). Naturally occurring hydrogen sulfide (H2S) works as a vasculoprotective gasotransmitter in vivo. However, given its reactive and hazardous nature, H2S is difficult to administer systemically. Here, we developed a hydrogel capable of localized slow release of precise amounts of H2S and tested its benefits on IH. The H2S-releasing hydrogel was prepared from a short peptide attached to an S-aroylthiooxime H2S donor. Upon dissolution in aqueous buffer, the peptide self-assembled into nanofibers, which formed a gel in the presence of calcium. This new hydrogel delivered H2S over the course of several hours, in contrast with fast-releasing NaHS. The H2S-releasing peptide/gel inhibited proliferation and migration of primary human vascular smooth muscle cells (VSMCs), while promoting proliferation and migration of human umbilical endothelial cells (ECs). Both NaHS and the H2S-releasing gel limited IH in human great saphenous vein segments obtained from vascular patients undergoing bypass surgery, with the H2S-releasing gel showing efficacy at a 5x lower dose than NaHS. These results suggest local perivascular H2S release as a new strategy to limit VSMC proliferation and IH while promoting EC proliferation, hence re-endothelialization. STATEMENT OF SIGNIFICANCE: Arterial occlusive disease is the leading cause of death in Western countries, yet current therapies suffer from high failure rates due to intimal hyperplasia (IH), a thickening of the vascular wall leading to secondary vessel occlusion. Hydrogen sulfide (H2S) is a gasotransmitter with vasculoprotective properties. Here we designed and synthesized a peptide-based H2S-releasing hydrogel and found that local application of the gel reduced IH in human vein segments obtained from patients undergoing bypass surgery. This work provides the first evidence of H2S efficacy against IH in human tissue, and the results show that the gel is more effective than NaHS, a common instantaneous H2S donor.

Cellular effects of AP102, a somatostatin analog with balanced affinities for the hSSTR2 and hSSTR5 receptors.

BACKGROUND: Somatostatin analogs (SSAs) are first-line medical therapy for the treatment of acromegaly and neuroendocrine tumors that express somatostatin receptors (SSTR). Somatostatin suppresses secretion of a large number of hormones through the stimulation of the five SSTR. However, unbalanced inhibition of secretion as observed with the highly potent SSAs pasireotide causes hyperglycaemia mainly by inhibiting insulin secretion. In contrast, AP102 a new SSAs has neutral effect on blood glucose while suppressing GH secretion. Our objective was to establish the cellular effects of AP102 on SSTR2 and SSTR5 that may explain the differences observed between AP102 and other SSAs. METHODS: We compared the binding and agonist activity of AP102 with somatostatin-14, octreotide and pasireotide in HEK293 cells transfected with human SSTR2 and SSTR5 receptors. SSAs signal transduction effects (cAMP concentrations) were measured in forskolin-treated cells in the presence of SSAs. Proliferation and apoptotic effects were determined and binding assays were performed using 125I- somatostatin-14. RESULTS: AP102 has comparable affinity and agonist effect to octreotide at SSTR2 (IC50's of 112 pM and 244 pM, respectively; EC50's of 230 pM and 210 pM, respectively) in contrast to pasireotide that exhibits a 12-27 fold higher IC50 (3110 pM) and about 5-fold higher EC50 (1097 pM). At SSTR5, AP102 has much higher affinity and stimulating effect than octreotide (IC50's of 773 pM and 16,737 pM, respectively; EC50's of 8526 pM and 26,800 pM), and an intermediate affinity and agonist effect between octreotide and pasireotide. AP102, octreotide and pasireotide have variable anti-proliferative effects on HEK cells transfected with SSTR2 and SSTR5. CONCLUSION: AP102 is a new SSA that better reduces signaling at SSTR2 than SSTR5 and prevents cell proliferation at both receptors. The euglycaemic effect of AP102 observed in preclinical studies may be related to this intermediate agonistic potency between pasireotide and octreotide at SSTR2 and SSTR5.

Dysfunctional autophagy following exposure to pro-inflammatory cytokines contributes to pancreatic ß-cell apoptosis.

Type 1 diabetes (T1D) results from ß-cell destruction due to concerted action of both innate and adaptive immune responses. Pro-inflammatory cytokines, such as interleukin-1ß and interferon-γ, secreted by the immune cells invading islets of Langerhans, contribute to pancreatic ß-cell death in T1D. Cytokine-induced endoplasmic reticulum (ER) stress plays a central role in ß-cell demise. ER stress can modulate autophagic response; however, no study addressed the regulation of autophagy during the pathophysiology of T1D. In this study, we document that cytokines activate the AMPK-ULK-1 pathway while inhibiting mTORC1, which stimulates autophagy activity in an ER stress-dependent manner. On the other hand, time-course analysis of LC3-II accumulation in autophagosomes revealed that cytokines block the autophagy flux in an ER stress independent manner, leading to the formation of large dysfunctional autophagosomes and worsening of ER stress. Cytokines rapidly impair lysosome function, leading to lysosome membrane permeabilization, Cathepsin B leakage and lysosomal cell death. Blocking cathepsin activity partially protects against cytokine-induced or torin1-induced apoptosis, whereas blocking autophagy aggravates cytokine-induced CHOP overexpression and ß-cell apoptosis. In conclusion, cytokines stimulate the early steps of autophagy while blocking the autophagic flux, which aggravate ER stress and trigger lysosomal cell death. Restoration of autophagy/lysosomal function may represent a novel strategy to improve ß-cell resistance in the context of T1D.

Targeting Cx40 (Connexin40) Expression or Function Reduces Angiogenesis in the Developing Mouse Retina.

OBJECTIVE: Cx40 (Connexin40) forms intercellular channels that coordinate the electric conduction in the heart and the vasomotor tone in large vessels. The protein was shown to regulate tumoral angiogenesis; however, whether Cx40 also contributes to physiological angiogenesis is still unknown. APPROACH AND RESULTS: Here, we show that Cx40 contributes to physiological angiogenesis. Genetic deletion of Cx40 leads to a reduction in vascular growth and capillary density in the neovascularization model of the mouse neonatal retina. At the angiogenic front, vessel sprouting is reduced, and the mural cells recruited along the sprouts display an altered phenotype. These alterations can be attributed to disturbed endothelial cell functions as selective reexpression of Cx40 in these cells restores normal angiogenesis. In vitro, targeting Cx40 in microvascular endothelial cells, by silencing its expression or by blocking gap junction channels, decreases their proliferation. Moreover, loss of Cx40 in these cells also increases their release of PDGF (platelet-derived growth factor) and promotes the chemoattraction of mural cells. In vivo, an intravitreal injection of a Cx40 inhibitory peptide, phenocopies the loss of Cx40 in the retinal vasculature of wild-type mice. CONCLUSIONS: Collectively, our data show that endothelial Cx40 contributes to the early stages of physiological angiogenesis in the developing retina, by regulating vessel growth and maturation. Cx40 thus represents a novel therapeutic target for treating pathological ocular angiogenesis.

Connexin37 reduces smooth muscle cell proliferation and intimal hyperplasia in a mouse model of carotid artery ligation.

AIMS: Intimal hyperplasia (IH) is an abnormal response to vessel injury characterized by the dedifferentiation, migration, and proliferation of quiescent vascular smooth muscle cells (VSMC) to form a neointima layer. Vascular connexins (Cx) are involved in the pathophysiology of various vascular diseases, and Cx43, the main Cx expressed in VSMC, has been shown to promote VSMC proliferation and IH. The aim of this study was to investigate the participation of another Cx, namely Cx37, in the formation of the neointima layer. METHODS AND RESULTS: Wild-type (WT) and Cx37-deficient (Cx37-/-) C57BL/6J mice were subjected to carotid artery ligation (CAL), a model of vessel injury and IH. The neointima developed linearly in WT until 28 days post surgery. In contrast, the neointima layer was almost absent 14 days after surgery in Cx37-/- mice, and twice as more developed after 28 days compared to WT mice. This large neointima formation correlated with a two-fold increase in cell proliferation in the media and neointima regions between 14 and 28 days in Cx37-/- mice compared to WT mice. The CAL triggered Cx43 overexpression in the media and neointima layers of ligated carotids in WT mice, and selectively up-regulated Cx37 expression in the media layer, but not in the neointima layer. The de novo expression of Cx37 in human primary VSMC reduced cell proliferation and P-Akt levels, in association with lower Cx43 levels, whereas Cx43 overexpression increased P-Akt levels. CONCLUSION: The presence of Cx37 in the media layer of injured arteries restrains VSMC proliferation and limits the development of IH, presumably by interfering with the pro-proliferative effect of Cx43 and the Akt pathway.

Store-operated Ca2+ entry: a key component of the insulin secretion machinery.

Normal plasma glucose level is ensured by the action of insulin, the major hypoglycemic hormone. Therefore, it is not surprising that insulin release from pancreatic ß-cells of the islets of Langerhans is controlled by an array of balanced mechanisms in which glucose plays the leading role. Glucose triggers insulin secretion through the well-described pathway of ATP-driven closure of ATP-sensitive potassium channels (KATP), depolarization of the plasma membrane, and opening of the voltage-dependent Ca2+ channels (VDCC). The subsequent rapid rise in cytoplasmic free Ca2+ concentration triggers insulin exocytosis. However, despite more than 40 years of investigation, certain aspects of the intracellular Ca2+ responses to glucose and secretagogues remain unexplained, suggesting the involvement of additional Ca2+ channels. Here, we discuss the emerging role of store-operated Ca2+ channels carried by Orai1 and transient receptor potential canonical 1 (TRPC1) proteins and regulated by the stromal interaction molecule 1 (STIM1) in the control of glucose-induced insulin secretion. The role of other voltage-independent cation channels formed by other members of the TRP channels family is also addressed.