Remco Haasdijk

Endothelial Cell–Specific FGD5 Involvement in Vascular Pruning Defines Neovessel Fate in Mice

Background— New vessel formation contributes to organ development during embryogenesis and tissue repair in response to mechanical damage, inflammation, and ischemia in adult organisms. Early angiogenesis includes formation of an excessive primitive network that needs to be reorganized into a secondary vascular network with higher hierarchical structure. Vascular pruning, the removal of aberrant neovessels by apoptosis, is a vital step in this process. Although multiple molecular pathways for early angiogenesis have been identified, little is known about the genetic regulators of secondary network development. Methods and Results— Using a transcriptomics approach, we identified a new endothelial specific gene named FYVE, RhoGEF, and PH domain–containing 5 (FGD5) that plays a crucial role in vascular pruning. Loss- and gain-of-function studies demonstrate that FGD5 inhibits neovascularization, indicated by in vitro tube-formation, aortic-ring, and coated-bead assays and by in vivo coated-bead plug assays and studies in the murine retina model. FGD5 promotes apoptosis-induced vaso-obliteration via induction of the hey1-p53 pathway by direct binding and activation of cdc42. Indeed, FGD5 correlates with apoptosis in endothelial cells during vascular remodeling and was linked to rising p21CIP1 levels in aging mice. Conclusion— We have identified FGD5 as a novel genetic regulator of vascular pruning by activation of endothelial cell–targeted apoptosis.

Apelin Enhances Cardiac Neovascularization After Myocardial Infarction by Recruiting Aplnr+ Circulating Cells

Rationale: Neovascularization stimulated by local or recruited stem cells after ischemia is a key process that salvages damaged tissue and shows similarities with embryonic vascularization. Apelin receptor (Aplnr) and its endogenous ligand apelin play an important role in cardiovascular development. However, the role of apelin signaling in stem cell recruitment after ischemia is unknown. Objective: To investigate the role of apelin signaling in recruitment after ischemia. Methods and Results: Aplnr was specifically expressed in circulating cKit+/Flk1+ cells but not in circulating Sca1+/Flk1+ and Lin+ cells. cKit+/Flk1+/Aplnr+ cells increased significantly early after myocardial ischemia but not after hind limb ischemia, indicative of an important role for apelin/Aplnr in cell recruitment during the nascent biological repair response after myocardial damage. In line with this finding, apelin expression was upregulated in the infarcted myocardium. Injection of apelin into the ischemic myocardium resulted in accelerated and increased recruitment of cKit+/Flk1+/Aplnr+ cells to the heart. Recruited Aplnr+/cKit+/Flk1+ cells promoted neovascularization in the peri-infarct area by paracrine activity rather than active transdifferentiation, resulting into cardioprotection as indicated by diminished scar formation and improved residual cardiac function. Aplnr knockdown in the bone marrow resulted in aggravation of myocardial ischemia–associated damage, which could not be rescued by apelin. Conclusions: We conclude that apelin functions as a new and potent chemoattractant for circulating cKit+/Flk1+/Aplnr+ cells during early myocardial repair, providing myocardial protection against ischemic damage by improving neovascularization via paracine action.

Cerebral cavernous malformations: from molecular pathogenesis to genetic counselling and clinical management

Cerebral cavernous (or capillary-venous) malformations (CCM) have a prevalence of about 0.1–0.5% in the general population. Genes mutated in CCM encode proteins that modulate junction formation between vascular endothelial cells. Mutations lead to the development of abnormal vascular structures.In this article, we review the clinical features, molecular and genetic basis of the disease, and management.

Ets2 Determines the Inflammatory State of Endothelial Cells in Advanced Atherosclerotic Lesions

Rationale: Neovascularization is required for embryonic development and plays a central role in diseases in adults. In atherosclerosis, the role of neovascularization remains to be elucidated. In a genome-wide microarray-screen of Flk1+ angioblasts during murine embryogenesis, the v-ets erythroblastosis virus E26 oncogene homolog 2 (Ets2) transcription factor was identified as a potential angiogenic factor. Objectives: We assessed the role of Ets2 in endothelial cells during atherosclerotic lesion progression toward plaque instability. Methods and Results: In 91 patients treated for carotid artery disease, Ets2 levels showed modest correlations with capillary growth, thrombogenicity, and rising levels of tumor necrosis factor-&agr; (TNF&agr;), monocyte chemoattractant protein 1, and interleukin-6 in the atherosclerotic lesions. Experiments in ApoE−/− mice, using a vulnerable plaque model, showed that Ets2 expression was increased under atherogenic conditions and was augmented specifically in the vulnerable versus stable lesions. In endothelial cell cultures, Ets2 expression and activation was responsive to the atherogenic cytokine TNF&agr;. In the murine vulnerable plaque model, overexpression of Ets2 promoted lesion growth with neovessel formation, hemorrhaging, and plaque destabilization. In contrast, Ets2 silencing, using a lentiviral shRNA construct, promoted lesion stabilization. In vitro studies showed that Ets2 was crucial for TNF&agr;-induced expression of monocyte chemoattractant protein 1, interleukin-6, and vascular cell adhesion molecule 1 in endothelial cells. In addition, Ets2 promoted tube formation and amplified TNF&agr;-induced loss of vascular endothelial integrity. Evaluation in a murine retina model further validated the role of Ets2 in regulating vessel inflammation and endothelial leakage. Conclusions: We provide the first evidence for the plaque-destabilizing role of Ets2 in atherosclerosis development by induction of an intraplaque proinflammatory phenotype in endothelial cells.

PDGF-Induced Migration of Vascular Smooth Muscle Cells Is Inhibited by Heme Oxygenase-1 Via VEGFR2 Upregulation and Subsequent Assembly of Inactive VEGFR2/PDGFRβ Heterodimers

Objective—In cardiovascular regulation, heme oxygenase-1 (HO-1) activity has been shown to inhibit vascular smooth muscle cell (VSMC) proliferation by promoting cell cycle arrest at the G1/S phase. However, the effect of HO-1 on VSMC migration remains unclear. We aim to elucidate the mechanism by which HO-1 regulates PDGFBB-induced VSMC migration. Methods and Results—Transduction of HO-1 cDNA adenoviral vector severely impeded human VSMC migration in a scratch, transmembrane, and directional migration assay in response to PDGFBB stimulation. Similarly, HO-1 overexpression in the remodeling process during murine retinal vasculature development attenuated VSMC coverage over the major arterial branches as compared with sham vector-transduced eyes. HO-1 expression in VSMCs significantly upregulated VEGFA and VEGFR2 expression, which subsequently promoted the formation of inactive PDGFR&bgr;/VEGFR2 complexes. This compromised PDGFR&bgr; phosphorylation and impeded the downstream cascade of FAK-p38 signaling. siRNA-mediated silencing of VEGFA or VEGFR2 could reverse the inhibitory effect of HO-1 on VSMC migration. Conclusion—These findings identify a potent antimigratory function of HO-1 in VSMCs, a mechanism that involves VEGFA and VEGFR2 upregulation, followed by assembly of inactive VEGFR2/PDGFR&bgr; complexes that attenuates effective PDGFR&bgr; signaling.

THSD1 preserves vascular integrity and protects against intraplaque haemorrhaging in ApoE−/− mice

AIMS Impairment of the endothelial barrier leads to microvascular breakdown in cardiovascular disease and is involved in intraplaque haemorrhaging and the progression of advanced atherosclerotic lesions that are vulnerable to rupture. The exact mechanism that regulates vascular integrity requires further definition. Using a microarray screen for angiogenesis-associated genes during murine embryogenesis, we identified thrombospondin type I domain 1 (THSD1) as a new putative angiopotent factor with unknown biological function. We sought to characterize the role of THSD1 in endothelial cells during vascular development and cardiovascular disease. METHODS AND RESULTS Functional knockdown of Thsd1 in zebrafish embryos and in a murine retina vascularization model induced severe haemorrhaging without affecting neovascular growth. In human carotid endarterectomy specimens, THSD1 expression by endothelial cells was detected in advanced atherosclerotic lesions with intraplaque haemorrhaging, but was absent in stable lesions, implying involvement of THSD1 in neovascular bleeding. In vitro, stimulation with pro-atherogenic factors (3% O2 and TNFα) decreased THSD1 expression in human endothelial cells, whereas stimulation with an anti-atherogenic factor (IL10) showed opposite effect. Therapeutic evaluation in a murine advanced atherosclerosis model showed that Thsd1 overexpression decreased plaque vulnerability by attenuating intraplaque vascular leakage, subsequently reducing macrophage accumulation and necrotic core size. Mechanistic studies in human endothelial cells demonstrated that THSD1 activates FAK-PI3K, leading to Rac1-mediated actin cytoskeleton regulation of adherens junctions and focal adhesion assembly. CONCLUSION THSD1 is a new regulator of endothelial barrier function during vascular development and protects intraplaque microvessels against haemorrhaging in advanced atherosclerotic lesions.