Notch ligand Delta-like 1 is essential for postnatal arteriogenesis

Growth of functional arteries is essential for the restoration of blood flow to ischemic organs. Notch signaling regulates arterial differentiation upstream of ephrin-B2 during embryonic development, but its role during postnatal arteriogenesis is unknown. Here, we identify the Notch ligand Delta-like 1 (Dll1) as an essential regulator of postnatal arteriogenesis. Dll1 expression was specifically detected in arterial endothelial cells, but not in venous endothelial cells or capillaries. During ischemia-induced arteriogenesis endothelial Dll1 expression was strongly induced, Notch signaling activated and ephrin-B2 upregulated, whereas perivascular cells expressed proangiogenic vascular endothelial growth factor, and the ephrin-B2 activator EphB4. In heterozygous Dll1 mutant mice endothelial Notch activation and ephrin-B2 induction after hindlimb ischemia were absent, arterial collateral growth was abrogated and recovery of blood flow was severely impaired, but perivascular vascular endothelial growth factor and EphB4 expression was unaltered. In vitro, angiogenic growth factors synergistically activated Notch signaling by induction of Dll1, which was necessary and sufficient to regulate ephrin-B2 expression and to induce ephrin-B2 and EphB4-dependent branching morphogenesis in human arterial EC. Thus, Dll1-mediated Notch activation regulates ephrin-B2 expression and postnatal arteriogenesis.     

Translation of PDGF cardioprotective pathways

Vascular function in the aging heart is impaired and may underlie the increased morbidity and mortality associated with ischemic heart disease in older individuals. This vascular dysfunction is due, in part, to impairment of plateletderived growth factor (PDGF)-mediated pathways in senescent cardiac microvascular endothelial cells. Restoration of these pathways by intramyocardial injection of growth factor transiently rescues senescent cardiac angiogenesis. Longer-term reconstitution can be achieved experimentally by transplantation of young bone marrow-derived stem cells to promote senescent cardiac angiogenic function in the murine host. Moreover, enhancement of PDGF pathways is cardioprotective, markedly reducing the extent of myocardial injury following coronary occlusion. The clinical translation of these findings for treatment of ischemic heart diseases must overcome the limitation of the proatherosclerotic actions of PDGF, as well as the generation of autologous stem/precursor cell approaches, for the aging cardiovascular system. Strategies targeting growth factor and/or stem-cell homing to gene products downstream of PDGF in the cardiac microvasculature may provide positive feedback loops to enhance cardiac angiogenesis and protection from myocardial infarction and may offer a foundation for developing novel therapies for the prevention and treatment of cardiovascular disease associated with aging.     

Impaired CXCR4 signaling contributes to the reduced neovascularization capacity of endothelial progenitor cells from patients with coronary artery disease

Transplantation of bone marrow cells as well as circulating endothelial progenitor cells (EPC) enhances neovascularization after ischemia. The chemokine receptor CXCR4 is essential for migration and homing of hematopoietic stem cells. Therefore, we investigated the role of CXCR4 and its downstream signaling cascade for the angiogenic capacity of cultured human EPC. Ex vivo, differentiated EPC derived from peripheral blood abundantly expressed CXCR4. Incubation of EPC from healthy volunteers with neutralizing antibodies against CXCR4 profoundly inhibited vascular endothelial growth factor- and stromal-derived factor-1-induced migration as well as EPC-induced angiogenesis in an ex vivo assay. Preincubation of transplanted EPC with CXCR4 antibody reduced EPC incorporation and impaired blood-flow recovery in ischemic hindlimbs of nude mice (57+/-4% of normal perfusion versus untreated EPC: 80+/-11%, P<0.001). Bone marrow mononuclear cells (BM-MNC) or EPC of heterozygous CXCR4(+/-) mice displayed reduced CXCR4 expression and disclosed impaired in vivo capacity to enhance recovery of ischemic blood flow in nude mice (blood flow 27+/-11% versus 66+/-25% using wild-type cells, P<0.01). Importantly, impaired blood flow in ischemic CXCR4(+/-) mice was rescued by injection of wild-type BM-MNC. Next, we investigated the role of CXCR4 for functional capacities of EPC from patients with coronary artery disease (CAD). Surface expression of CXCR4 was similar in EPC from patients with CAD compared with healthy controls. However, basal Janus kinase (JAK)-2 phosphorylation was significantly reduced and less responsive to stromal-derived factor-1 in EPC from patients with CAD compared with healthy volunteers, indicating that CXCR4-mediated JAK-2 signaling is dysregulated in EPC from patients with CAD. The CXCR4 receptor signaling profoundly modulates the angiogenic activity and homing capacity of cultured human EPC. Disturbance of CXCR4 signaling, as demonstrated by reduced JAK-2 phosphorylation, may contribute to functional impairment of EPC from patients with CAD. Stimulating CXCR4 signaling might improve functional properties of EPC and may rescue impaired neovascularization capacity of EPC derived from patients with CAD.     

Fibroblast growth factor regulation of neovascularization

PURPOSE OF REVIEW: Fibroblast growth factors are potent angiogenic inducers; however, their precise roles in angiogenesis have not been well understood. In this review, we will focus on specific roles played by fibroblast growth factors in neovascularization. RECENT FINDINGS: Although fibroblast growth factors promote a strong angiogenic response, it has been suggested that FGF-induced angiogenesis requires activation of the vascular endothelial growth factor system. Recent findings have endorsed the view of indirect contribution of fibroblast growth factor signaling to vascular development. A study using embryoid bodies demonstrated a nonimmediate role played by fibrobalst growth factor receptor 1 in vasculogenesis as vascular endothelial growth factor supplementation was sufficient to promote vascular development in Fgfr1-/- embryoid bodies. Moreover, another line of evidence indicated that myocardial fibroblast growth factor signaling is essential for mouse coronary development. The key role of fibroblast growth factor signaling in this process is Hedgehog activation, which induces vascular endothelial growth factor expression and formation of the coronary vasculature.In addition to vascular endothelial growth factor interaction, fibroblast growth factors can control neovascularization by influencing other growth factors and chemokines such as platelet-derived growth factor, hepatocyte growth factor and monocyte chemoattractant protein-1, contributing to development of mature vessels and collateral arteries. SUMMARY: Although fibroblast growth factors are potent angiogenic factors, they may indirectly control neovascularization in concert with other growth factors. Thus, the unique role played by fibroblast growth factors might be organization of various angiogenic pathways and coordination of cell-cell interactions in this process.     

Restoration of coronary collateral growth in the Zucker obese rat:

The metabolic syndrome (MS), a condition characterized by several risk factors for coronary artery disease, including obesity, is associated with endothelial dysfunction and oxidative stress. Because proper endothelial function is essential for signaling of certain growth factors (vascular endothelial growth factor, VEGF) we hypothesized that coronary collateral growth (CCG) is impaired in a model of the MS. To test this hypothesis, we stimulated coronary collateral growth in pre-diabetic Zucker obese fatty rats (OZR) and lean littermates (LZR) by using episodic, repetitive ischemia (RI: 40 s left anterior descending arterial occlusion, 24/d for 14 d). Myocardial blood flow (MBF, radioactive microspheres) was measured in the normal (NZ) and collateral-dependent (ischemic) zones (CZ); CCG was assessed as a ratio of CZ/NZ flow (unity represents complete restoration of CZ flow). In LZR, CZ/NZ ratio increased from 0.18 ± 0.03 to 0.81 ± 0.07 after RI (P < 0.05). In contrast, in OZR rats CZ/NZ did not increase after RI (0.15 ± 0.04 vs 0.18 ± 0.04). To rectify abrogated collateral growth in OZR, we employed VEGF gene therapy (VEGF-transduced, strained-matched, cultured vascular smooth muscle cells [cVSMCs], delivered intracoronary). VEGF therapy modestly but not significantly increased the CZ/NZ ratio after RI (0.16 ± 0.05 vs 0.33 ± 0.06). To facilitate VEGF signaling,we reduced oxidative stress by transducing cVSMCs with both ecSOD and VEGF. This increased the CZ/NZ flow ratio after RI to 0.52 ± 0.04 (p < 0.05 vs. OZR [(0.19 ± 0.04]) indicating partial restoration of collateral growth. Our results demonstrate that coronary collateral growth is impaired in a model of the metabolic syndrome and that growth factor gene therapy with VEGF is made far more effective when it is coupled to an intervention that reduces oxidative stress. Supported by NIH grants HL32788 (WMC), HL65203 (WMC), HL73755 (WMC), and COBRE RR018766 (FP, PR,WMC), and American Heart Association Scientist Development Grant (PR), AHA Postdoctoral Fellowship Award (NH, PR).     

Loss of bcl-2 during the senescence exacerbates the impaired angiogenic functions in endothelial cells by deteriorating the mitochondrial redox state

Ageing is an important risk factor for ischemic cardiovascular diseases, although its underlying molecular mechanisms remain to be elucidated. Here, we report a crucial role of Bcl-2 in the impaired angiogenic functions in senescent endothelial cells (ECs) by modulating the mitochondrial redox state. Cellular senescence impaired angiogenic functions in ECs without attenuating the mitogen-activated protein kinase or Akt signaling, and vascular endothelial growth factor receptor 2 or Tie-2 expressions. We identified that Bcl-2 expression was markedly reduced in 3 independent models for senescent ECs, and pharmacological inhibition, as well as small interfering RNA-mediated gene silencing of Bcl-2, significantly impaired the angiogenic functions in young ECs. Bcl-2 has an antioxidative role by locating the glutathione at mitochondria, and we found that mitochondrial oxidative stress was significantly augmented in senescent ECs, in association with reduced mitochondria-associated glutathione. Transfection of Bcl-2 in senescent ECs significantly reduced the mitochondrial oxidative stress, restored the mitochondrial membrane potential, and improved the angiogenic capacity. Furthermore, gene transfer of Bcl-2 using adenovirus significantly improved the in vivo angiogenesis in the Matrigel plugs implanted into aged mice, whereas the Bcl-2 inhibitor reduced the angiogenesis in the Matrigel plugs implanted into young mice. Together, Bcl-2 plays a crucial role in the regulation of the mitochondrial redox state in ECs, and, thus, loss of Bcl-2 during the senescence exacerbates the impaired angiogenesis by augmenting the mitochondrial oxidative stress.     

The emerging role of IGF-1 deficiency in cardiovascular aging: recent advances

This review focuses on cardiovascular protective effects of insulin-like growth factor (IGF)-1, provides a landscape of molecular mechanisms involved in cardiovascular alterations in patients and animal models with congenital and adult-onset IGF-1 deficiency, and explores the link between age-related IGF-1 deficiency and the molecular, cellular, and functional changes that occur in the cardiovascular system during aging. Microvascular protection conferred by endocrine and paracrine IGF-1 signaling, its implications for the pathophysiology of cardiac failure and vascular cognitive impairment, and the role of impaired cellular stress resistance in cardiovascular aging considered here are based on emerging knowledge of the effects of IGF-1 on Nrf2-driven antioxidant response.     

Young adult bone marrow-derived endothelial precursor cells restore aging-impaired cardiac angiogenic function

Delivery of young bone marrow-derived stem cells offers a novel approach for restoring the impaired senescent cardiac angiogenic function that may underlie the increased morbidity and mortality associated with ischemic heart disease in older individuals. Recently, we reported that alterations in endothelial cells of the aging heart lead to a dysregulation in the cardiac myocyte platelet-derived growth factor (PDGF)-B-induced paracrine pathway, which contributes to impaired cardiac angiogenic function. Based on these results, we hypothesized that cellular restoration of the PDGF pathway by bone marrow-derived endothelial precursor cells (EPCs) could reverse the aging-associated decline in angiogenic activity. In vitro studies revealed that young murine (3-month-old) bone marrow-derived EPCs recapitulated the cardiac myocyte-induced expression of PDGF-B, whereas EPCs from the bone marrow of aging mice (18-month-old) did not express PDGF-B when cultured in the presence of cardiac myocytes. Transplantation of young, but not old, genetically marked syngeneic bone marrow cells into intact, unirradiated aging mice that populated the endogenous senescent murine bone marrow incorporated into the neovasculature of subsequently transplanted syngeneic neonatal myocardium. Moreover, the young bone marrow-derived EPCs restored the senescent host angiogenic PDGF-B induction pathway and cardiac angiogenesis, with graft survival and myocardial activity in the aging murine host (cardiac allograft viability: 3-month-old controls, 8/8; 18-month-old controls, 1/8; 18-month-old donors receiving bone marrow from 3-month-old mice, 15/16; or 18-month-old mice, 0/6; P<0.05). These results may offer a foundation for the development of novel therapies for the prevention and treatment of cardiovascular disease associated with aging.     

Liver-specific knockdown of IGF-1 decreases vascular oxidative stress resistance by impairing the Nrf2-dependent antioxidant response: a novel model of vascular aging

Recent studies demonstrate that age-related dysfunction of NF-E2-related factor-2 (Nrf2)-driven pathways impairs cellular redox homeostasis, exacerbating age-related cellular oxidative stress and increasing sensitivity of aged vessels to oxidative stress-induced cellular damage. Circulating levels of insulin-like growth factor (IGF)-1 decline during aging, which significantly increases the risk for cardiovascular diseases in humans. To test the hypothesis that adult-onset IGF-1 deficiency impairs Nrf2-driven pathways in the vasculature, we utilized a novel mouse model with a liver-specific adeno-associated viral knockdown of the Igf1 gene using Cre-lox technology (Igf1(f/f) + MUP-iCre-AAV8), which exhibits a significant decrease in circulating IGF-1 levels (~50%). In the aortas of IGF-1-deficient mice, there was a trend for decreased expression of Nrf2 and the Nrf2 target genes GCLC, NQO1 and HMOX1. In cultured aorta segments of IGF-1-deficient mice treated with oxidative stressors (high glucose, oxidized low-density lipoprotein, and H(2)O(2)), induction of Nrf2-driven genes was significantly attenuated as compared with control vessels, which was associated with an exacerbation of endothelial dysfunction, increased oxidative stress, and apoptosis, mimicking the aging phenotype. In conclusion, endocrine IGF-1 deficiency is associated with dysregulation of Nrf2-dependent antioxidant responses in the vasculature, which likely promotes an adverse vascular phenotype under pathophysiological conditions associated with oxidative stress (eg, diabetes mellitus, hypertension) and results in accelerated vascular impairments in aging.     

Central Arterial Aging and Angiotensin II Signaling

Arterial remodeling over time is a cornerstone of normal systemic aging. The age-associated arterial structural and functional changes in the intima, the media, and the adventitia are closely linked to angiotensin II (Ang II) signaling. A growing line of evidence indicates that essential elements of Ang II signaling, which encompasses milk fat globule epidermal growth factor-8, calpain-1, transforming growth factor-β1, matrix metalloproteinase-2/9, monocyte chemoattractant protein-1, nicotinamide adenine dinucleotide phosphate-oxidase, and reactive oxygen species, are upregulated within the central arterial wall in rats, nonhuman primates, and humans during aging. In vitro studies show that the elevation of Ang II signaling induces the accumulation of collagen and advanced glycated end-products, the degradation of elastin, and the increased cell cycle disorder, invasion, and hypertrophy of endothelial and vascular smooth muscle cells. Further, in vivo studies demonstrate that increased Ang II signaling accelerates arterial aging. Conversely, attenuating Ang II signaling via an inhibition of angiotensin conversing enzyme or a blockade of AT1 activation retards age-associated arterial remodeling. This review attempts to integrate complex facts of Ang II signaling within the aged central arterial wall and may shed light on new therapeutic targets for arterial aging.     

Epithelial expression of angiogenic growth factors modulate arterial vasculogenesis in human liver development

Intrahepatic bile ducts maintain a close anatomical relationship with hepatic arteries. During liver ontogenesis, the development of the hepatic artery appears to be modulated by unknown signals originating from the bile duct. Given the capability of cholangiocytes to produce angiogenic growth factors and influence peribiliary vascularization, we studied the immunohistochemical expression of vascular endothelial growth factor (VEGF), angiopoietin-1, angiopoietin-2, and their cognate receptors (VEGFR-1, VEGFR-2, Tie-2) in fetal human livers at different gestational ages and in mice characterized by defective biliary morphogenesis (Hnf6(-/-)). The results showed that throughout the different developmental stages, VEGF was expressed by developing bile ducts and angiopoietin-1 by hepatoblasts, whereas their cognate receptors were variably expressed by vascular cells according to the different maturational stages. Precursors of endothelial and mural cells expressed VEGFR-2 and Tie-2, respectively. In immature hepatic arteries, endothelial cells expressed VEGFR-1, whereas mural cells expressed both Tie-2 and Angiopoietin-2. In mature hepatic arteries, endothelial cells expressed Tie-2 along with VEGFR-1. In early postnatal Hnf6(-/-) mice, VEGF-expressing ductal plates failed to incorporate into the portal mesenchyma, resulting in severely altered arterial vasculogenesis. CONCLUSION: The reciprocal expression of angiogenic growth factors and receptors during development supports their involvement in the cross talk between liver epithelial cells and the portal vasculature. Cholangiocytes generate a VEGF gradient that is crucial during the migratory stage, when it determines arterial vasculogenesis in their vicinity, whereas angiopoietin-1 signaling from hepatoblasts contributes to the remodeling of the hepatic artery necessary to meet the demands of the developing epithelium.     

Smoking in Asians: it doesn't stop at vascular endothelium

Smoking is a global risk factor for atherosclerosis, affecting societies all over the world. Smoking exerts its pro-atherogenic effects by triggering the generation of free radicals and by modifying vascular redox signaling. These abnormal vascular responses to cigarette smoking result into impaired endothelial function, decreased nitric oxide bioavailability, increased intima media thickness and finally atherosclerotic plaque formation in human arteries. Importantly, evidence suggests that cigarette smoking may have an effect on vascular smooth muscle cells function, leading to impaired endothelium-independent dilation in response to nitrate, in the brachial artery of healthy smokers. Taken together, it is now well established that smoking induces functional and structural abnormalities in the vascular wall, by mechanisms involving endothelial dysfunction and impairment of vascular smooth muscle cells in human arterial tree.     

EGFL7: a unique angiogenic signaling factor in vascular development and disease

EGFL7 is a secreted angiogenic factor that is highly conserved in vertebrates. Most secreted angiogenic signaling molecules, including VEGF and fibroblast growth factor-2, are mainly expressed by non-endothelial cell types such as fibroblasts. In contrast, EGFL7 is unique because it is almost exclusively expressed by and acts on endothelial cells. Egfl7 expression is highest when the endothelium is in an active, proliferating state. This factor acts as a chemoattractant for endothelial cells and binds to components of the extracellular matrix. In vivo, Egfl7 is important for regulating tubulogenesis in zebrafish and for controlling vascular patterning and integrity in mice. Its function in blood vessel development is mediated, at least in part, through modulation of Notch signaling. In this review, we summarize the findings that support a role for Egfl7 in developmental and postnatal angiogenesis and describe the EGFL7-signaling pathways that underlie these processes. In addition, we discuss a potential role for EGFL7 in vascular repair and its possible use as a therapeutic target for treatment of hypoxia-induced injury. Finally, we consider EGFL7 action during tumorigenesis and its potential as an antiangiogenic agent.