Int J Mol Sci

The role of Hedgehog (Hh) signaling in vascular biology has first been highlighted in embryos by Pepicelli et al. in 1998 and Rowitch et al. in 1999. Since then, the proangiogenic role of the Hh ligands has been confirmed in adults, especially under pathologic conditions. More recently, the Hh signaling has been proposed to improve vascular integrity especially at the blood-brain barrier (BBB). However, molecular and cellular mechanisms underlying the role of the Hh signaling in vascular biology remain poorly understood and conflicting results have been reported. As a matter of fact, in several settings, it is currently not clear whether Hh ligands promote vessel integrity and quiescence or destabilize vessels to promote angiogenesis. The present review relates the current knowledge regarding the role of the Hh signaling in vasculature development, maturation and maintenance, discusses the underlying proposed mechanisms and highlights controversial data which may serve as a guideline for future research. Most importantly, fully understanding such mechanisms is critical for the development of safe and efficient therapies to target the Hh signaling in both cancer and cardiovascular/cerebrovascular diseases

Observations on the perfusion recovery of regenerative angiogenesis in an ischemic limb model under hyperoxia

This study combines two well-known vascular research models, hyperoxia and hind limb ischemia, aiming to better characterize capacities of the hyperoxia challenge. We studied two groups of C57/BL6 male mice, a control (C) and a hind limb ischemia (HLI) group. Perfusion from both limbs was recorded in all animals by laser Doppler techniques under an oxygen (O2 ) saturated atmosphere, once for control and, during 35 days for the HLI group. We used a third set of normoxic animals for HLI morphometric control. The expected variability of responses was higher for the younger animals. In the HLI group, capillary density normalized at Day 21 as expected, but not microcirculatory physiology. In the operated limb, perfusion decreased dramatically following surgery (Day 4), as a slight reduction in the non-operated limb was also noted. Consistently, the response to hyperoxia was an increased perfusion in the ischemic limb and decreased perfusion in the contralateral limb. Only at Day 35, both limbs exhibited similar flows, although noticeably lower than Day 0. These observations help to understand some of the functional variability attributed to the hyperoxia model, by showing (i) differences in the circulation of the limb pairs to readjust a new perfusion set-point even after ischemia, an original finding implying that (ii) data from both limbs should be recorded when performing distal measurements in vivo. Our data demonstrate that the new vessels following HLI are not functionally normal, and this also affects the non-operated limb. These findings confirm the discriminative capacities of the hyperoxia challenge and suggest its potential utility to study other pathologies with vascular impact

Arterioscler Thromb Vasc Biol

Evidences accumulated within the past decades identified hedgehog signaling as a new regulator of endothelium integrity. More specifically, we recently identified Dhh (desert hedgehog) as a downstream effector of Klf2 (Kruppel-like factor 2) in endothelial cells (ECs). The purpose of this study is to investigate whether hedgehog coreceptors Gas1 (growth arrest-specific 1) and Cdon (cell adhesion molecule-related/downregulated by oncogenes) may be used as therapeutic targets to modulate Dhh signaling in ECs. Approach and Results: We demonstrated that both Gas1 and Cdon are expressed in adult ECs and relied on either siRNAs- or EC-specific conditional knockout mice to investigate their role. We found that Gas1 deficiency mainly phenocopies Dhh deficiency especially by inducing VCAM-1 (vascular cell adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1) overexpression while Cdon deficiency has opposite effects by promoting endothelial junction integrity. At a molecular level, Cdon prevents Dhh binding to Ptch1 (patched-1) and thus acts as a decoy receptor for Dhh, while Gas1 promotes Dhh binding to Smo (smoothened) and as a result potentiates Dhh effects. Since Cdon is upregulated in ECs treated by inflammatory cytokines, including TNF (tumor necrosis factor)-α and Il (interleukin)-1β, we then tested whether Cdon inhibition would promote endothelium integrity in acute inflammatory conditions and found that both fibrinogen and IgG extravasation were decreased in association with an increased Cdh5 (cadherin-5) expression in the brain cortex of EC-specific Cdon knockout mice administered locally with Il-1β. Altogether, these results demonstrate that Gas1 is a positive regulator of Dhh in ECs while Cdon is a negative regulator. Interestingly, Cdon blocking molecules may then be used to promote endothelium integrity, at least in inflammatory conditions

J Thromb Haemost

Essentials To reliably study the respective roles of blood and endothelial cells in hemostasis, mouse models with a strong and specific endothelial expression of the Cre recombinase are needed. Using mT/mG reporter mice and conditional JAK2 mice, we compared Pdgfb-iCreERT2 and Cdh5(PAC)-CreERT2 with well-characterized Tie2-Cre mice. Comparison of recombination efficiency and specificity towards blood lineage reveals major differences between endothelial transgenic mice. Cre-mediated recombination occurs in a small number of adult hematopoietic stem cells in Pdgfb-iCreERT2;JAK2 transgenic mice. SUMMARY: Background The vessel wall, and particularly blood endothelial cells (BECs), are intensively studied to better understand hemostasis and target thrombosis. To understand the specific role of BECs, it is important to have mouse models that allow specific and homogeneous expression of genes of interest in all BEC beds without concomitant expression in blood cells. Inducible Pdgfb-iCreERT2 and Cdh5(PAC)-CreERT2 transgenic mice are widely used for BEC targeting. However, issues remain in terms of recombination efficiency and specificity regarding hematopoietic cells. Objectives To determine which mouse model to choose when strong expression of a transgene is required in adult BECs from various organs, without concomitant expression in hematopoietic cells. Methods Using mT/mG reporter mice to measure recombination efficiency and conditional JAK2 mice to assess specificity regarding hematopoietic cells, we compared Pdgfb-iCreERT2 and Cdh5(PAC)-CreERT2 with well-characterized Tie2-Cre mice. Results Adult Cdh5(PAC)-CreERT2 mice are endothelial specific but require a dose of 10 mg of tamoxifen to allow constant Cre expression. Pdgfb-iCreERT2 mice injected with 5 mg of tamoxifen are appropriate for most endothelial research fields except liver studies, as hepatic sinusoid ECs are not recombined. Surprisingly, 2 months after induction of Cre-mediated recombination, all Pdgfb-iCreERT2;JAK2 mice developed a myeloproliferative neoplasm that is related to the presence of JAK2V617F in hematopoietic cells, showing for the first time that Cre-mediated recombination occurs in a small number of adult hematopoietic stem cells in Pdgfb-iCreERT2 transgenic mice. Conclusion This study provides useful guidelines for choosing the best mouse line to study the role of BECs in hemostasis and thrombosis

PLoS Biol

Inflammation of the central nervous system (CNS) induces endothelial blood-brain barrier (BBB) opening as well as the formation of a tight junction barrier between reactive astrocytes at the Glia Limitans. We hypothesized that the CNS parenchyma may acquire protection from the reactive astrocytic Glia Limitans not only during neuroinflammation but also when BBB integrity is compromised in the resting state. Previous studies found that astrocyte-derived Sonic hedgehog (SHH) stabilizes the BBB during CNS inflammatory disease, while endothelial-derived desert hedgehog (DHH) is expressed at the BBB under resting conditions. Here, we investigated the effects of endothelial Dhh on the integrity of the BBB and Glia Limitans. We first characterized DHH expression within endothelial cells at the BBB, then demonstrated that DHH is down-regulated during experimental autoimmune encephalomyelitis (EAE). Using a mouse model in which endothelial Dhh is inducibly deleted, we found that endothelial Dhh both opens the BBB via the modulation of forkhead box O1 (FoxO1) transcriptional activity and induces a tight junctional barrier at the Glia Limitans. We confirmed the relevance of this glial barrier system in human multiple sclerosis active lesions. These results provide evidence for the novel concept of "chronic neuroinflammatory tolerance" in which BBB opening in the resting state is sufficient to stimulate a protective barrier at the Glia Limitans that limits the severity of subsequent neuroinflammatory disease. In summary, genetic disruption of the BBB generates endothelial signals that drive the formation under resting conditions of a secondary barrier at the Glia Limitans with protective effects against subsequent CNS inflammation. The concept of a reciprocally regulated CNS double barrier system has implications for treatment strategies in both the acute and chronic phases of multiple sclerosis pathophysiology

Vascular endothelial cell expression of JAK2V617F is sufficient to promote a pro-thrombotic state due to increased P-selectin expression

Thrombosis is the main cause of morbidity and mortality in patients with JAK2V617F myeloproliferative neoplasms. Recent studies have reported the presence of JAK2V617F in endothelial cells of some patients with myeloproliferative neoplasms. We investigated the role of endothelial cells that express JAK2V617F in thrombus formation using an in vitro model of human endothelial cells overexpressing JAK2V617F and an in vivo model of mice with endothelial-specific JAK2V617F expression. Interestingly, these mice displayed a higher propensity for thrombus. When deciphering the mechanisms by which JAK2V617F-expressing endothelial cells promote thrombosis, we observed that they have a pro-adhesive phenotype associated with increased endothelial P-selectin exposure, secondary to degranulation of Weibel-Palade bodies. We demonstrated that P-selectin blockade was sufficient to reduce the increased propensity of thrombosis. Moreover, treatment with hydroxyurea also reduced thrombosis and decreased the pathological interaction between leukocytes and JAK2V617F-expressing endothelial cells through direct reduction of endothelial P-selectin expression. Taken together, our data provide evidence that JAK2V617F-expressing endothelial cells promote thrombosis through induction of endothelial P-selectin expression, which can be reversed by hydroxyurea. Our findings increase our understanding of thrombosis in patients with myeloproliferative neoplasms, at least those with JAK2V617F-positive endothelial cells, and highlight a new role for hydroxyurea. This novel finding provides the proof of concept that an acquired genetic mutation can affect the pro-thrombotic nature of endothelial cells, suggesting that other mutations in endothelial cells could be causal in thrombotic disorders of unknown cause, which account for 50% of recurrent venous thromboses

Sur un nouveau mode de traitement du coryza spasmodique avec ou sans hydrorrhée par les injections interstitielles de paraffine

Thèse : Médecine : Université de Bordeaux : 1906N° d'ordre : 7

Role of Hedgehog Signaling in Vasculature Development, Differentiation, and Maintenance

International audienceThe role of Hedgehog (Hh) signaling in vascular biology has first been highlighted in embryos by Pepicelli et al. in 1998 and Rowitch et al. in 1999. Since then, the proangiogenic role of the Hh ligands has been confirmed in adults, especially under pathologic conditions. More recently, the Hh signaling has been proposed to improve vascular integrity especially at the blood-brain barrier (BBB). However, molecular and cellular mechanisms underlying the role of the Hh signaling in vascular biology remain poorly understood and conflicting results have been reported. As a matter of fact, in several settings, it is currently not clear whether Hh ligands promote vessel integrity and quiescence or destabilize vessels to promote angiogenesis. The present review relates the current knowledge regarding the role of the Hh signaling in vasculature development, maturation and maintenance, discusses the underlying proposed mechanisms and highlights controversial data which may serve as a guideline for future research. Most importantly, fully understanding such mechanisms is critical for the development of safe and efficient therapies to target the Hh signaling in both cancer and cardiovascular/cerebrovascular diseases

Endothelial Dysfunction in Heart Failure With Preserved Ejection Fraction: What are the Experimental Proofs?

Heart failure with preserved ejection fraction (HFpEF) has been recognized as the greatest single unmet need in cardiovascular medicine. Indeed, the morbi-mortality of HFpEF is high and as the population ages and the comorbidities increase, so considerably does the prevalence of HFpEF. However, HFpEF pathophysiology is still poorly understood and therapeutic targets are missing. An unifying, but untested, theory of the pathophysiology of HFpEF, proposed in 2013, suggests that cardiovascular risk factors lead to a systemic inflammation, which triggers endothelial cells (EC) and coronary microvascular dysfunction. This cardiac small vessel disease is proposed to be responsible for cardiac wall stiffening and diastolic dysfunction. This paradigm is based on the fact that microvascular dysfunction is highly prevalent in HFpEF patients. More specifically, HFpEF patients have been shown to have decreased cardiac microvascular density, systemic endothelial dysfunction and a lower mean coronary flow reserve. Importantly, impaired coronary microvascular function has been associated with the severity of HF. This review discusses evidence supporting the causal role of endothelial dysfunction in the pathophysiology of HFpEF in human and experimental models

Endogenous Sonic Hedgehog limits inflammation and angiogenesis in the ischaemic skeletal muscle of mice

Aims: Hedgehog (Hh) signalling has been shown to be re-activated in ischaemic tissues and participate in ischaemia-induced angiogenesis. Sonic Hedgehog (Shh) is upregulated by more than 80-fold in the ischaemic skeletal muscle, however its specific role in ischaemia-induced angiogenesis has not yet been fully investigated. The purpose of the present study was to investigate the role of endogenous Shh in ischaemia-induced angiogenesis. Methods and results: To this aim, we used inducible Shh knock-out (KO) mice and unexpectedly found that capillary density was significantly increased in re-generating muscle of Shh deficient mice 5 days after hind limb ischaemia was induced, demonstrating that endogenous Shh does not promote angiogenesis but more likely limits it. Myosin and MyoD expression were equivalent in Shh deficient mice and control mice, indicating that endogenous Shh is not required for ischaemia-induced myogenesis. Additionally, we observed a significant increase in macrophage infiltration in the ischaemic muscle of Shh deficient mice. Our data indicate that this was due to an increase in chemokine expression by myoblasts in the setting of impaired Hh signalling, using tissue specific Smoothened conditional KO mice. The increased macrophage infiltration in mice deficient for Hh signalling in myocytes was associated with increased VEGFA expression and a transiently increased angiogenesis, demonstrating that Shh limits inflammation and angiogenesis indirectly by signalling to myocytes. Conclusion: Although ectopic administration of Shh has previously been shown to promote ischaemia-induced angiogenesis, the present study reveals that endogenous Shh does not promote ischaemia-induced angiogenesis. On the contrary, the absence of Shh leads to aberrant ischaemic tissue inflammation and a transiently increased angiogenesis