Ayman Al Haj Zen

Human Adult Vena Saphena Contains Perivascular Progenitor Cells Endowed With Clonogenic and Proangiogenic Potential

Background— Clinical trials in ischemic patients showed the safety and benefit of autologous bone marrow progenitor cell transplantation. Non–bone marrow progenitor cells with proangiogenic capacities have been described, yet they remain clinically unexploited owing to their scarcity, difficulty of access, and low ex vivo expansibility. We investigated the presence, antigenic profile, expansion capacity, and proangiogenic potential of progenitor cells from the saphenous vein of patients undergoing coronary artery bypass surgery. Methods and Results— CD34-positive cells, negative for the endothelial marker von Willebrand factor, were localized around adventitial vasa vasorum. After dissection of the vein from surrounding tissues and enzymatic digestion, CD34-positive/CD31-negative cells were isolated by selective culture, immunomagnetic beads, or fluorescence-assisted cell sorting. In the presence of serum, CD34-positive/CD31-negative cells gave rise to a highly proliferative population that expressed pericyte/mesenchymal antigens together with the stem cell marker Sox2 and showed clonogenic and multilineage differentiation capacities. We called this population “saphenous vein–derived progenitor cells” (SVPs). In culture, SVPs integrated into networks formed by endothelial cells and supported angiogenesis through paracrine mechanisms. Reciprocally, endothelial cell–released factors facilitated SVP migration. These interactive responses were inhibited by Tie-2 or platelet-derived growth factor-BB blockade. Intramuscular injection of SVPs in ischemic limbs of immunodeficient mice improved neovascularization and blood flow recovery. At 14 days after transplantation, proliferating SVPs were still detectable in the recipient muscles, where they established N-cadherin–mediated physical contact with the capillary endothelium. Conclusions— SVPs generated from human vein CD34-positive/CD31-negative progenitor cells might represent a new therapeutic tool for angiogenic therapy in ischemic patients.

Inhibition of Delta-Like-4–Mediated Signaling Impairs Reparative Angiogenesis After Ischemia

Rationale: Notch signaling regulates vascular development. However, the implication of the Notch ligand Delta-like 4 (Dll4) in postischemic angiogenesis remains unclear. Objective: We investigated the role of Dll4/Notch signaling in reparative angiogenesis using a mouse model of ischemia. Methods and Results: We found Dll4 weakly expressed in microvascular endothelial cells of normoperfused muscles. Conversely, Dll4 is upregulated following ischemia and localized at the forefront of sprouting capillaries. We analyzed the effect of inhibiting endogenous Dll4 by intramuscular injection of an adenovirus encoding the soluble form of Dll4 extracellular domain (Ad-sDll4). Dll4 inhibition caused the formation of a disorganized, low-perfused capillary network in ischemic muscles. This structural abnormality was associated to delayed blood flow recovery and muscle hypoxia and degeneration. Analysis of microvasculature at early stages of repair revealed that Dll4 inhibition enhances capillary sprouting in a chaotic fashion and causes excessive leukocyte infiltration of ischemic muscles. Furthermore, Dll4 inhibition potentiated the elevation of the leukocyte chemoattractant CXCL1 (chemokine [C-X-C motif] ligand 1) following ischemia, without altering peripheral blood levels of stromal cell–derived factor-1 and monocyte chemoattractant protein-1. In cultured human monocytes, Dll4 induces the transcription of Notch target gene Hes-1 and inhibits the basal and tumor necrosis factor-&agr;–stimulated production of interleukin-8, the human functional homolog of murine CXCL1. The inhibitory effect of Dll4 on interleukin-8 was abolished by DAPT, a Notch inhibitor, or by coculturing activated human monocytes with Ad-sDll4–infected endothelial cells. Conclusions: Dll4/Notch interaction is essential for proper reparative angiogenesis. Moreover, Dll4/Notch signaling regulates sprouting angiogenesis and coordinates the interaction between inflammation and angiogenesis under ischemic conditions.

Reduced Immunoregulatory CD31+ T Cells in the Blood of Atherosclerotic Mice With Plaque Thrombosis

Objective—Lymphocyte activation is thought to play a major role in the pathogenesis of atherosclerotic complications such as plaque thrombosis. Circulating CD31+ T cells have been shown to regulate human T cell activation. Aim of this study was to evaluate whether the proportion of circulating immunoregulatory CD31+ T cells is correlated to the occurrence of plaque thrombosis in aged apolipoprotein (apo) E knockout (KO) mice. Methods and Results—CD31+ T cell depletion of spleen T cells enhanced proliferation (P<0.05) and interferon-γ production (P<0.01) while reducing interleukin (IL)-4 (P<0.001) and IL-10 (P=0.001) secretion in response to minimally modified low-density lipoprotein. CD31+ T cells were counted in 65 apoE KO mice (46-week-old) by flow cytometry. Organizing thrombi could be documented in 28 of 195 (14%) lesions and in at least one of the aorta root lesions in 23 of 65 mice (35%). CD31+ T cell count was significantly reduced in mice showing plaque thrombosis (72.3±1.5% versus 84.1±1.2%; P<0.0001), but such reduction did not follow induced plaque rupture or experimentally controlled thrombosis. Conclusions—Reduced CD31+ T cells in circulating blood is a hallmark of atherosclerotic plaque thrombosis. Our data suggest that CD31+ T cells may play an important regulatory role in the development of plaque thrombosis.

Notch signalling in ischaemia-induced angiogenesis

Notch signalling represents a key pathway essential for normal vascular development. Recently, great attention has been focused on the implication of Notch pathway components in postnatal angiogenesis and regenerative medicine. This paper critically reviews the most recent findings supporting the role of Notch in ischaemia-induced neovascularization. Notch signalling reportedly regulates several steps of the reparative process occurring in ischaemic tissues, including sprouting angiogenesis, vessel maturation, interaction of vascular cells with recruited leucocytes and skeletal myocyte regeneration. Further characterization of Notch interaction with other signalling pathways might help identify novel targets for therapeutic angiogenesis.

The Retinoid Agonist Tazarotene Promotes Angiogenesis and Wound Healing

Therapeutic angiogenesis is a major goal of regenerative medicine, but no clinically approved small molecule exists that enhances new blood vessel formation. Here we show, using a phenotype-driven high-content imaging screen of an annotated chemical library of 1,280 bioactive small molecules, that the retinoid agonist Tazarotene, enhances in vitro angiogenesis, promoting branching morphogenesis, and tubule remodeling. The proangiogenic phenotype is mediated by retinoic acid receptor but not retinoic X receptor activation, and is characterized by secretion of the proangiogenic factors hepatocyte growth factor, vascular endothelial growth factor, plasminogen activator, urokinase and placental growth factor, and reduced secretion of the antiangiogenic factor pentraxin-3 from adjacent fibroblasts. In vivo, Tazarotene enhanced the growth of mature and functional microvessels in Matrigel implants and wound healing models, and increased blood flow. Notably, in ear punch wound healing model, Tazarotene promoted tissue repair characterized by rapid ear punch closure with normal-appearing skin containing new hair follicles, and maturing collagen fibers. Our study suggests that Tazarotene, an FDA-approved small molecule, could be potentially exploited for therapeutic applications in neovascularization and wound healing.

Bone morphogenetic protein and Notch signalling crosstalk in poor-prognosis, mesenchymal-subtype colorectal cancer

The functional role of bone morphogenetic protein (BMP) signalling in colorectal cancer (CRC) is poorly defined, with contradictory results in cancer cell line models reflecting the inherent difficulties of assessing a signalling pathway that is context‐dependent and subject to genetic constraints. By assessing the transcriptional response of a diploid human colonic epithelial cell line to BMP ligand stimulation, we generated a prognostic BMP signalling signature, which was applied to multiple CRC datasets to investigate BMP heterogeneity across CRC molecular subtypes. We linked BMP and Notch signalling pathway activity and function in human colonic epithelial cells, and normal and neoplastic tissue. BMP induced Notch through a γ‐secretase‐independent interaction, regulated by the SMAD proteins. In homeostasis, BMP/Notch co‐localization was restricted to cells at the top of the intestinal crypt, with more widespread interaction in some human CRC samples. BMP signalling was downregulated in the majority of CRCs, but was conserved specifically in mesenchymal‐subtype tumours, where it interacts with Notch to induce an epithelial–mesenchymal transition (EMT) phenotype. In intestinal homeostasis, BMP–Notch pathway crosstalk is restricted to differentiating cells through stringent pathway segregation. Conserved BMP activity and loss of signalling stringency in mesenchymal‐subtype tumours promotes a synergistic BMP–Notch interaction, and this correlates with poor patient prognosis. BMP signalling heterogeneity across CRC subtypes and cell lines can account for previous experimental contradictions. Crosstalk between the BMP and Notch pathways will render mesenchymal‐subtype CRC insensitive to γ‐secretase inhibition unless BMP activation is concomitantly addressed.

BMP and Notch interaction in CRC subtypes

The functional role of bone morphogenetic protein (BMP) signalling in colorectal cancer (CRC) is poorly defined, with contradictory results in cancer cell line models reflecting the inherent difficulties of assessing a signalling pathway that is context‐dependent and subject to genetic constraints. By assessing the transcriptional response of a diploid human colonic epithelial cell line to BMP ligand stimulation, we generated a prognostic BMP signalling signature, which was applied to multiple CRC datasets to investigate BMP heterogeneity across CRC molecular subtypes. We linked BMP and Notch signalling pathway activity and function in human colonic epithelial cells, and normal and neoplastic tissue. BMP induced Notch through a γ‐secretase‐independent interaction, regulated by the SMAD proteins. In homeostasis, BMP/Notch co‐localization was restricted to cells at the top of the intestinal crypt, with more widespread interaction in some human CRC samples. BMP signalling was downregulated in the majority of CRCs, but was conserved specifically in mesenchymal‐subtype tumours, where it interacts with Notch to induce an epithelial–mesenchymal transition (EMT) phenotype. In intestinal homeostasis, BMP–Notch pathway crosstalk is restricted to differentiating cells through stringent pathway segregation. Conserved BMP activity and loss of signalling stringency in mesenchymal‐subtype tumours promotes a synergistic BMP–Notch interaction, and this correlates with poor patient prognosis. BMP signalling heterogeneity across CRC subtypes and cell lines can account for previous experimental contradictions. Crosstalk between the BMP and Notch pathways will render mesenchymal‐subtype CRC insensitive to γ‐secretase inhibition unless BMP activation is concomitantly addressed.

DLK1: a novel negative regulator of angiogenesis?

This editorial refers to ‘The non-canonical NOTCH ligand DLK1 exhibits a novel vascular role as a strong inhibitor of angiogenesis’ by P. Rodriguez et al. , pp. 232–241, this issue. The Notch signalling pathway is pivotal in normal vascular development and post-natal angiogenesis. Cumulative evidence supports the involvement of three canonical Notch ligands—Delta-like 1 (Dll1), Delta-like 4 (Dll4), and Jagged-1 (Jag1)—in the angiogenesis process. They are believed to function as Notch signalling agonists/antagonists to regulate the position and behaviour of endothelial cells under activated conditions. For instance, tissue-derived vascular endothelial growth factor (VEGF) induces the expression of the Dll4 ligand on endothelial tip cells, which in turn activates Notch signalling in neighbouring endothelial ‘stalk’ cells and restricts the emergence of excessive sprouting through repression of VEGF receptors and consequent reduction in responsiveness to VEGF.1–4 Moreover, Jag1 reportedly acts as an antagonist of the Dll4/Notch signalling in stalk cells, helping them maintain their activated phenotype.5 In this context, the coordination of different components of the Notch signalling seems to be a key determinant for proper and functional neovascularization.6,7 …

MicroRNA-148b Targets the TGF-β Pathway to Regulate Angiogenesis and Endothelial-to-Mesenchymal Transition during Skin Wound Healing

Transforming growth factor beta (TGF-β) is crucial for regulation of the endothelial cell (EC) homeostasis. Perturbation of TGF-β signaling leads to pathological conditions in the vasculature, causing cardiovascular disease and fibrotic disorders. The TGF-β pathway is critical in endothelial-to-mesenchymal transition (EndMT), but a gap remains in our understanding of the regulation of TGF-β and related signaling in the endothelium. This study applied a gain- and loss-of function approach and an in vivo model of skin wound healing to demonstrate that miR-148b regulates TGF-β signaling and has a key role in EndMT, targeting TGFB2 and SMAD2. Overexpression of miR-148b increased EC migration, proliferation, and angiogenesis, whereas its inhibition promoted EndMT. Cytokine challenge decreased miR-148b levels in ECs while promoting EndMT through the regulation of SMAD2. Finally, in a mouse model of skin wound healing, delivery of miR-148b mimics promoted wound vascularization and accelerated closure. In contrast, inhibition of miR-148b enhanced EndMT in wounds, resulting in impaired wound closure that was reversed by SMAD2 silencing. Together, these results demonstrate for the first time that miR-148b is a key factor controlling EndMT and vascularization. This opens new avenues for therapeutic application of miR-148b in vascular and tissue repair.

Phenotypic miRNA Screen Identifies miR-26b to Promote the Growth and Survival of Endothelial Cells

Endothelial cell (EC) proliferation is a crucial event in physiological and pathological angiogenesis. MicroRNAs (miRNAs) have emerged as important modulators of the angiogenic switch. Here we conducted high-content screening of a human miRNA mimic library to identify novel regulators of EC growth systematically. Several miRNAs were nominated that enhanced or inhibited EC growth. Of these, we focused on miR-26b, which is a conserved candidate and expressed in multiple human EC types. miR-26b overexpression enhanced EC proliferation, migration, and tube formation, while inhibition of miR-26b suppressed the proliferative and angiogenic capacity of ECs. A combinatory functional small interfering RNA (siRNA) screening of 48 predicted gene targets revealed that miR-26b enhanced EC growth and survival through inhibiting PTEN expression. Local administration of miR-26b mimics promoted the growth of new microvessels in the Matrigel plug model. In the mouse model of hindlimb ischemia, miR-26b was found to be downregulated in endothelium in the first week following ischemia, and local overexpression of miR-26b improved the survival of capillaries and muscle fibers in ischemic muscles. Our findings suggest that miR-26b enhances EC proliferation, survival, and angiogenesis. miR-26b is a potential target for developing novel pro-angiogenic therapeutics in ischemic disease.