Search

Background: Lower-limb peripheral artery disease is one of the major complications of diabetes. Peripheral artery disease is associated with poor limb and cardiovascular prognoses, along with a dramatic decrease in life expectancy. Despite major medical advances in the treatment of diabetes, a substantial therapeutic gap remains in the peripheral artery disease population. Praliciguat is an orally available sGC (soluble guanylate cyclase) stimulator that has been reported both preclinically and in early stage clinical trials to have favorable effects in metabolic and hemodynamic outcomes, suggesting that it may have a potential beneficial effect in peripheral artery disease., Methods: We evaluated the effect of praliciguat on hind limb ischemia recovery in a mouse model of type 2 diabetes. Hind limb ischemia was induced in leptin receptor-deficient (Lepr db/db ) mice by ligation and excision of the left femoral artery. Praliciguat (10 mg/kg/day) was administered in the diet starting 3 days before surgery., Results: Twenty-eight days after surgery, ischemic foot perfusion and function parameters were better in praliciguat-treated mice than in vehicle controls. Improved ischemic foot perfusion was not associated with either improved traditional cardiovascular risk factors (ie, weight, glycemia) or increased angiogenesis. However, treatment with praliciguat significantly increased arteriole diameter, decreased ICAM1 (intercellular adhesion molecule 1) expression, and prevented the accumulation of oxidative proangiogenic and proinflammatory muscle fibers. While investigating the mechanism underlying the beneficial effects of praliciguat therapy, we found that praliciguat significantly downregulated Myh2 and Cxcl12 mRNA expression in cultured myoblasts and that conditioned medium form praliciguat-treated myoblast decreased ICAM1 mRNA expression in endothelial cells. These results suggest that praliciguat therapy may decrease ICAM1 expression in endothelial cells by downregulating Cxcl12 in myocytes., Conclusions: Our results demonstrated that praliciguat promotes blood flow recovery in the ischemic muscle of mice with type 2 diabetes, at least in part by increasing arteriole diameter and by downregulating ICAM1 expression.

Rationale: Tamoxifen prevents the recurrence of breast cancer and is also beneficial against bone demineralization and arterial diseases. It acts as an ER (estrogen receptor) α antagonist in ER-positive breast cancers, whereas it mimics the protective action of 17β-estradiol in other tissues such as arteries. However, the mechanisms of these tissue-specific actions remain unclear., Objective: Here, we tested whether tamoxifen is able to accelerate endothelial healing and analyzed the underlying mechanisms., Methods and Results: Using 3 complementary mouse models of carotid artery injury, we demonstrated that both tamoxifen and estradiol accelerated endothelial healing, but only tamoxifen required the presence of the underlying medial smooth muscle cells. Chronic treatment with 17β-estradiol and tamoxifen elicited differential gene expression profiles in the carotid artery. The use of transgenic mouse models targeting either whole ERα in a cell-specific manner or ERα subfunctions (membrane/extranuclear versus genomic/transcriptional) demonstrated that 17β-estradiol-induced acceleration of endothelial healing is mediated by membrane ERα in endothelial cells, while the effect of tamoxifen is mediated by the nuclear actions of ERα in smooth muscle cells., Conclusions: Whereas tamoxifen acts as an antiestrogen and ERα antagonist in breast cancer but also on the membrane ERα of endothelial cells, it accelerates endothelial healing through activation of nuclear ERα in smooth muscle cells, inviting to revisit the mechanisms of action of selective modulation of ERα.

Rationale: Klf (kruppel-like factor) 2 is critical to establish and maintain endothelial integrity., Objective: Therefore, determining upstream and downstream mediators of Klf2 would lead to alternative therapeutic targets in cardiovascular disease management., Methods and Results: Here we identify Dhh (desert hedgehog) as a downstream effector of Klf2, whose expression in endothelial cells (ECs) is upregulated by shear stress and decreased by inflammatory cytokines. Consequently, we show that Dhh knockdown in ECs promotes endothelial permeability and EC activation and that Dhh agonist prevents TNF-α (tumor necrosis factor alpha) or glucose-induced EC dysfunction. Moreover, we demonstrate that human critical limb ischemia, a pathological condition linked to diabetes mellitus and inflammation, is associated to major EC dysfunction. By recreating a complex model of critical limb ischemia in diabetic mice, we found that Dhh-signaling agonist significantly improved EC function without promoting angiogenesis, which subsequently improved muscle perfusion., Conclusion: Restoring EC function leads to significant critical limb ischemia recovery. Dhh appears to be a promising target, downstream of Klf2, to prevent the endothelial dysfunction involved in ischemic vascular diseases.

Rationale: A better understanding of the mechanism underlying skeletal muscle repair is required to develop therapies that promote tissue regeneration in adults. Hedgehog signaling has been shown previously to be involved in myogenesis and angiogenesis: 2 crucial processes for muscle development and regeneration., Objective: The objective of this study was to identify the role of the hedgehog transcription factor Gli3 in the cross-talk between angiogenesis and myogenesis in adults., Methods and Results: Using conditional knockout mice, we found that Gli3 deficiency in endothelial cells did not affect ischemic muscle repair, whereas in myocytes, Gli3 deficiency resulted in severely delayed ischemia-induced myogenesis. Moreover, angiogenesis was also significantly impaired in HSA-Cre(ERT2); Gli3(Flox/Flox) mice, demonstrating that impaired myogenesis indirectly affects ischemia-induced angiogenesis. The role of Gli3 in myocytes was then further investigated. We found that Gli3 promotes myoblast differentiation through myogenic factor 5 regulation. In addition, we found that Gli3 regulates several proangiogenic factors, including thymidine phosphorylase and angiopoietin-1 both in vitro and in vivo, which indirectly promote endothelial cell proliferation and arteriole formation. In addition, we found that Gli3 is upregulated in proliferating myoblasts by the cell cycle-associated transcription factor E2F1., Conclusions: This study shows for the first time that Gli3-regulated postnatal myogenesis is necessary for muscle repair-associated angiogenesis. Most importantly, it implies that myogenesis drives angiogenesis in the setting of skeletal muscle repair and identifies Gli3 as a potential target for regenerative medicine.

Rationale: Blood vessel growth and patterning have been shown to be regulated by nerve-derived signals. Desert hedgehog (Dhh), one of the Hedgehog family members, is expressed by Schwann cells of peripheral nerves., Objective: The purpose of this study was to investigate the contribution of Dhh to angiogenesis in the setting of ischemia., Methods and Results: We induced hindlimb ischemia in wild-type and Dhh(-/-) mice. First, we found that limb perfusion is significantly impaired in the absence of Dhh. This effect is associated with a significant decrease in capillary and artery density in Dhh(-/-). By using mice in which the Hedgehog signaling pathway effector Smoothened was specifically invalidated in endothelial cells, we demonstrated that Dhh does not promote angiogenesis by a direct activation of endothelial cells. On the contrary, we found that Dhh promotes peripheral nerve survival in the ischemic muscle and, by doing so, maintains the pool of nerve-derived proangiogenic factors. Consistently, we found that denervation of the leg, immediately after the onset of ischemia, severely impairs ischemia-induced angiogenesis and decreases expression of vascular endothelial growth factor A, angiopoietin 1, and neurotrophin 3 in the ischemic muscle., Conclusions: This study demonstrates the crucial roles of nerves and factors regulating nerve physiology in the setting of ischemia-induced angiogenesis.

The transcription factor cAMP responsive element-binding protein (CREB) has been found to be involved in arterial smooth muscle cell (SMC) migration. We previously demonstrated that osteopontin (OPN) expression is a key step for UTP-mediated migration of arterial SMCs and that activator protein (AP)-1, nuclear factor kappaB, and upstream stimulatory transcription factors are involved in this OPN expression. The present study aims to determine the role of CREB in UTP-induced migration and OPN expression in cultured SMCs. We found that CREB is activated by UTP via extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase pathways but not by protein kinase A. Both overexpression of a dominant negative CREB and CREB small interfering RNA treatment suppressed UTP-induced OPN expression and SMC migration. Gel-shift and chromatin immunoprecipitation assays revealed that CREB binds 2 AP-1 sites (-1870 and -76) and a cAMP responsive element-like site (-1403) on the OPN promoter. Mutations of these sites showed that only the 2 AP-1 sites were required for UTP-induced OPN expression. Moreover, gel-supershift and sequential chromatin immunoprecipitation assays suggested that CREB was associated with c-Fos on the AP-1 sites of the OPN promoter. These results demonstrate that CREB participates in the induction of UTP-activated OPN expression via its binding to 2 AP-1 sites and is thus involved in UTP-mediated SMC migration.

Osteopontin (OPN), an RGD-containing extracellular matrix protein, is associated with arterial smooth muscle cell (SMC) activation in vitro and in vivo. Many cytokines and growth factors involved in vessel wall remodeling induce OPN overexpression. Moreover, we recently demonstrated that the extracellular nucleotide UTP also induces OPN expression and that OPN is essential for UTP-mediated SMC migration. Thus, we set out to investigate the mechanisms of OPN expression. The aim of this study was to identify transcription factors involved in the regulation of OPN expression in SMCs. First, we explored the contribution of mRNA stabilization and transcription in the increase of UTP-induced OPN mRNA levels. We show that UTP induced OPN mRNA increases via both OPN mRNA stabilization and OPN promoter activation. Then, to identify transcription factors involved in UTP-induced OPN transcription, we located a promoter element activated by UTP within the rat OPN promoter using a gene reporter assay strategy. The -96 to +1 region mediated UTP-induced OPN overexpression (+276+/-60%). Sequence analysis of this region revealed a potential site for AP-1 located at -76. When this AP-1 site was deleted, UTP-induced activation of the -96 to +1 region was totally inhibited. Thus, this AP-1 (-76) site is involved in UTP-induced OPN transcription. A supershift assay revealed that both c-Fos and c-Jun bind to this AP-1 site. Finally, we demonstrate that angiotensin II and platelet-derived growth factor, two main factors involved in vessel wall pathology, also modulated OPN expression via AP-1 activation.

Many factors have been shown to be involved in the development of hyperplasic lesions of vessels, but the role of extracellular nucleotides remains largely unknown. The presence of P2Y and P2X nucleotide receptors on arterial endothelial and smooth muscle cells suggests a potential role for nucleotides in the vessel pathophysiology. Although the role of P2X in physiology of vessels is well documented, that of P2Y is not completely understood. We recently demonstrated that extracellular nucleotides, and particularly UTP, induced migration of cultured arterial smooth muscle cells (ASMCs). This migration is dependent on osteopontin expression and involves the Rho and mitogen-activated protein (MAP) kinase pathways. An important question is to determine the specific role of the different P2Y receptors of rat ASMCs in the UTP-induced migration process. Therefore, we first quantified mRNA levels of P2Y(2), P2Y(4), and P2Y(6) nucleotide receptors in cultured rat ASMCs by a competitive RT-PCR approach and demonstrated that P2Y(2) is the most highly expressed among these receptors potentially involved in the UTP-mediated response. In addition to UTP, UDP also induced ASMC migration even when UTP regeneration was inhibited, suggesting the involvement of UDP receptor P2Y(6). Moreover, suramin, a specific antagonist of rat P2Y(2) receptor, acted as an inhibitor of UTP-induced migration. Taken together, these results suggest a prominent role for the UTP receptor, P2Y(2), and for the UDP receptor, P2Y(6), in UTP-induced rat ASMC migration.

Migration and proliferation of arterial smooth muscle cells (SMCs) play a prominent role in the development of atherosclerotic plaques and restenosis lesions. Most of the growth-regulatory molecules potentially involved in these pathological conditions also demonstrate chemotactic properties. Extracellular purine and pyrimidine nucleotides have been shown to induce cell cycle progression and to elicit growth of cultured vascular SMCs. Moreover, the P2Y(2) ATP/UTP receptor was overexpressed in intimal thickening, suggesting a role of these nucleotides in vascular remodeling. Using the Transwell system migration assay, we demonstrate that extracellular ATP, UTP, and UDP exhibit a concentration-dependent chemotactic effect on cultured rat aortic SMCs. UTP, the most powerful nucleotide inducer of migration, elicited significant responses from 10 nmol/L. In parallel, UTP increased osteopontin expression dose-dependently. The blockade of osteopontin or its integrin receptors alpha(v)beta(3)/beta(5) by specific antibodies or antagonists inhibited UTP-induced migration. Moreover, the blockade of ERK-1/ERK-2 MAP kinase or rho protein pathways led to the inhibition of both UTP-induced osteopontin increase and migration, demonstrating the central role of osteopontin in this process. Taken together, these results suggest that extracellular nucleotides, and particularly UTP, can induce arterial SMC migration via the action of osteopontin.

Apoptosis of arterial smooth muscle cells (ASMCs) could play an important role in the pathogenesis of atherosclerosis and restenosis. Recent studies have demonstrated that extracellular adenosine induces apoptosis in various cell types. Our aim was to delineate the capacity of this nucleoside to induce ASMC apoptosis in arterial diseases. We demonstrate that adenosine dose-dependently triggers apoptosis of cultured human ASMCs. Apoptotic cell death was quantified by analysis of nuclear chromatin morphology and characterized by DNA laddering. The involvement of adenosine receptors was suggested, because neither an adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride, nor an inhibitor of cellular nucleoside transport, dipyridamole, was able to inhibit adenosine-induced ASMC apoptosis. In contrast, an A(1)/A(2)-adenosine receptor antagonist, xanthine amine congener, totally inhibited adenosine-induced apoptosis. Furthermore, among more selective inhibitors of P(1) purinoceptor subtypes, only alloxazine, an antagonist of A(1)- and A(2)-adenosine receptors, completely inhibited adenosine-induced ASMC apoptosis, suggesting that adenosine triggers ASMC apoptosis via either 1 or both of these receptors. However, 8-cyclopentyl-1,3-dipropylxanthine, 8-(3-chlorostyryl) caffeine, and 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1, 4-(+/-)-dihydropyridine-3,5-dicarboxylate, which are A(1)-, A(2a)-, and A(3)-adenosine receptor antagonists, did not inhibit adenosine-induced apoptosis, suggesting an involvement of the A(2b)-receptor in this process. Moreover, the cAMP increase followed by cAMP-dependent protein kinase activation appears essential to mediate adenosine-induced ASMC apoptosis, thus confirming the previous hypothesis. These results indicate that adenosine-induced apoptosis of ASMCs is essentially mediated via A(2b)-adenosine receptor and involves a cAMP-dependent pathway.