Abstract

The use of nanotechnology for medical purposes - nanomedicine - has grown exponentially over the past few decades. This is exemplified by the US Food and Drug Administration's approval of several nanotherapies for various conditions, as well as the funding of nanomedical programmes worldwide. Although originally the domain of anticancer therapy, recent advances have illustrated the considerable potential of nanomedicine in the diagnosis and treatment of atherosclerosis. This Review elaborates on nanoparticle-targeting concepts in atherosclerotic disease, provides an overview of the use of nanomedicine in atherosclerosis, and discusses potential future applications and clinical benefits.

Abstract

BACKGROUND:

Diabetic retinopathy and retinopathy of prematurity are diseases caused by pathological angiogenesis in the retina as a consequence of local hypoxia. The underlying mechanism for epiretinal neovascularization (tuft formation), which contributes to blindness, has yet to be identified. Neural cell adhesion molecule (N-CAM) is expressed by Müller cells and astrocytes, which are in close contact with the retinal vasculature, during normal developmental angiogenesis.

METHODOLOGY/PRINCIPAL FINDINGS:

Notably, during oxygen induced retinopathy (OIR) N-CAM accumulated on astrocytes surrounding the epiretinal tufts. Here, we show that N-CAM ablation results in reduced vascular tuft formation due to reduced endothelial cell proliferation despite an elevation in VEGFA mRNA expression, whereas retinal developmental angiogenesis was unaffected.

CONCLUSION/SIGNIFICANCE:

We conclude that N-CAM exhibits a regulatory function in pathological angiogenesis in OIR. This is a novel finding that can be of clinical relevance in diseases associated with proliferative vasculopathy.

Abstract

Following focal cerebral ischemia, blood vessels in the ischemic border, or penumbra, launch an angiogenic response. In light of the critical role for fibronectin in angiogenesis, and the observation that fibronectin and its integrin receptors are strongly upregulated on angiogenic vessels in the hypoxic CNS, the aim of this study was to establish whether angiogenic vessels in the ischemic CNS also show this response. Focal cerebral ischemia was established in C57/Bl6 mice by middle cerebral artery occlusion (MCA:O), and brain tissue analyzed 7days following re-perfusion, a time at which angiogenesis is ongoing. Within the ischemic core, immunofluorescent (IF) studies demonstrated vascular expression of MECA-32, a marker of leaky cerebral vessels, and vascular breakdown, defined by loss of staining for the endothelial marker, CD31, and the vascular adhesion molecules, laminin, dystroglycan and α6 integrin. Within the ischemic penumbra, dual-IF with CD31 and Ki67 revealed the presence of proliferating endothelial cells, indicating ongoing angiogenesis. Significantly, vessels in the ischemic penumbra showed strong upregulation of fibronectin and the fibronectin receptors, α5β1 and αvβ3 integrins. Taken together with our recent finding that the α5β1 integrin plays an important role in promoting cerebral angiogenesis in response to hypoxia, these results suggest that stimulation of the fibronectin-α5β1 integrin signaling pathway may provide a novel approach to amplifying the intrinsic angiogenic response to cerebral ischemia.

Abstract

The mouse retina is vascularized after birth when angiogenic blood vessels grow and sprout along a pre-formed latticework of astrocytes. How astrocyte-derived cues control patterns of blood vessel growth and sprouting, however, remains enigmatic. Here, we have used molecular genetic strategies in mice to demonstrate that αvβ8 integrin expressed in astrocytes is essential for neovascularization of the developing retina. Selective ablation of αv or β8 integrin gene expression in astrocytes leads to impaired blood vessel sprouting and intraretinal hemorrhage, particularly during formation of the secondary vascular plexus. These pathologies correlate, in part, with diminished αvβ8 integrin-mediated activation of extracellular matrix-bound latent transforming growth factor βs (TGFβs) and defective TGFβ signaling in vascular endothelial cells, but not astrocytes. Collectively, our data demonstrate that αvβ8 integrin is a component of a paracrine signaling axis that links astrocytes to blood vessels and is essential for proper regulation of retinal angiogenesis

Abstract

Blood vessels form extensive networks that nurture all tissues in the body. Abnormal vessel growth and function are hallmarks of cancer and ischemic and inflammatory diseases, and they contribute to disease progression. Therapeutic approaches to block vascular supply have reached the clinic, but limited efficacy and resistance pose unresolved challenges. Recent insights establish how endothelial cells communicate with each other and with their environment to form a branched vascular network. The emerging principles of vascular growth provide exciting new perspectives, the translation of which might overcome the current limitations of pro- and antiangiogenic medicine.

Abstract

Vascular Endothelial Growth Factor Receptor-2 (VEGFR2) is the major mediator of the angiogenic effects of VEGF. In addition to its well known role as a membrane receptor that activates multiple signaling pathways, VEGFR2 also has a nuclear localization. However, what VEGFR2 does in the nucleus is still unknown. In the present report we show that, in endothelial cells, nuclear VEGFR2 interacts with several nuclear proteins, including the Sp1, a transcription factor that has been implicated in the regulation of genes needed for angiogenesis. By in vivo chromatin immunoprecipitation (ChIP) assays, we found that VEGFR2 binds to the Sp1-responsive region of the VEGFR2 proximal promoter. These results were confirmed by EMSA assays, using the same region of the VEGFR2 promoter. Importantly, we show that the VEGFR2 DNA binding is directly linked to the transcriptional activation of the VEGFR2 promoter. By reporter assays, we found that the region between -300/-116 relative to the transcription start site is essential to confer VEGFR2-dependent transcriptional activity. It was previously described that nuclear translocation of the VEGFR2 is dependent on its activation by VEGF. In agreement, we observed that the binding of VEGFR2 to DNA requires VEGF activation, being blocked by Bevacizumab and Sunitinib, two anti-angiogenic agents that inhibit VEGFR2 activation. Our findings demonstrate a new mechanism by which VEGFR2 activates its own promoter that could be involved in amplifying the angiogenic response.

Abstract

How individual components of the vascular basement membrane influence endothelial cell behaviour remains unclear. Here we show that laminin α4 (Lama4) regulates tip cell numbers and vascular density by inducing endothelial Dll4/Notch signalling in vivo. Lama4 deficiency leads to reduced Dll4 expression, excessive filopodia and tip cell formation in the mouse retina, phenocopying the effects of Dll4/Notch inhibition. Lama4-mediated Dll4 expression requires a combination of integrins in vitro and integrin β1 in vivo. We conclude that appropriate laminin/integrin-induced signalling is necessary to induce physiologically functional levels of Dll4 expression and regulate branching frequency during sprouting angiogenesis in vivo.

Abstract

Angiogenesis inhibiting agents are currently integral component of anticancer therapy. However, tumors, initially responsive to anti-angiogenic drugs or vascular targeting agents, can acquire resistance. The limited clinical efficacy might result from the heterogeneous nature of tumors or alternatively from the unique phenotype of tumor vascular cells, widely diverse from so-called 'normal' endothelium. Hence, defining the molecular mechanisms driving this diversity might provide a rational basis to design combinatory therapies that should be more effective in avoiding resistance. Herein, we demonstrated that tumor-derived endothelial cells (TECs) isolated from breast and kidney carcinomas retained an endothelial phenotype, but outspread independently of growth factors. Applying small interfering RNA approach, we demonstrated that interleukin (IL)-3, but not vascular endothelial growth factor, released by TECs, supports their autocrine growth and promotes in vivo vessel formation and tumor angiogenesis. Meanwhile, we found that the expression of the membrane-bound kit ligand (mbKitL) depends on IL-3, and it is crucial for adhesion of endothelial progenitor cells (EPCs) and inflammatory cells to TECs. These events required Akt activation. Finally, the finding that depletion of the mbKitL prevented EPC and inflammatory cell trafficking into vascular microenvironment, indicates that, as in bone marrow, the mbKitL can act as a membrane/adhesion molecule for c-Kit-expressing cells. These data provide evidences that an IL-3 autocrine loop can drive a tumor endothelial switch and that targeting IL-3 might confer a significant therapeutic advantage to hamper tumor angiogenesis.Oncogene advance online publication, 6 June 2011; doi:10.1038/onc.2011.204.

Abstract

Dysregulation of angiogenesis is implicated in many diseases. Von Willebrand factor (VWF), a large plasma glycoprotein essential for normal haemostasis is synthesised by endothelial cells (EC) and megakaryocytes. Raised VWF plasma levels are a risk factor for arterial thrombosis, while deficiency of VWF causes Von Willebrand disease (VWD), the most common congenital bleeding disorder in man. VWD can be associated with angiodysplasia, vascular malformations linked to defective angiogenesis. We hypothesised that VWF is involved in angiogenesis. To test this hypothesis, we isolated mononuclear cells from peripheral blood of controls and patients with VWD and cultured them to obtain confluent monolayers of blood outgrowth endothelial cells (BOEC). BOEC from VWD patients showed decreased VWF release, consistent with the patients' clinical data, increased capillary tube formation on Matrigel, migration and proliferation compared to controls. Thus BOEC from VWD patients exhibit enhanced angiogenic properties. Increased angiogenesis was also observed after inhibition of VWF expression in human umbilical vein EC (HUVEC) with specific siRNA. Mechanism studies on VWF siRNA-treated HUVEC implicated the endothelial VWF receptor, integrin α V β3 and the angiogenesis regulator angiopoietin-2. To validate our findings in an in vivo model we studied the VWF-deficient mouse. In vivo Matrigel angiogenesis and imaging of blood vessels in the ear showed increased angiogenesis and vascular network compared to littermate controls. Thus we have identified a novel mechanism for the regulation of angiogenesis and a new function for VWF, which may have clinical implications for VWD and for cardiovascular disease.

Abstract

Vascular anastomosis is the cornerstone of vascular, cardiovascular and transplant surgery. Most anastomoses are performed with sutures, which are technically challenging and can lead to failure from intimal hyperplasia and foreign body reaction. Numerous alternatives to sutures have been proposed, but none has proven superior, particularly in small or atherosclerotic vessels. We have developed a new method of sutureless and atraumatic vascular anastomosis that uses US Food and Drug Administration (FDA)-approved thermoreversible tri-block polymers to temporarily maintain an open lumen for precise approximation with commercially available glues. We performed end-to-end anastomoses five times more rapidly than we performed hand-sewn controls, and vessels that were too small (<1.0 mm) to sew were successfully reconstructed with this sutureless approach. Imaging of reconstructed rat aorta confirmed equivalent patency, flow and burst strength, and histological analysis demonstrated decreased inflammation and fibrosis at up to 2 years after the procedure. This new technology has potential for improving efficiency and outcomes in the surgical treatment of cardiovascular disease.

Abstract

Rationale:Neovascularization favors intraplaque hemorrhage and plaque rupture. Development of therapeutic strategies against atheromatous angiogenesis requires elucidation of its initiating factors.Objective:We investigated the contribution of smooth muscle cells (SMCs) and atheroma-derived lipids to the initiation of atheroma-associated neoangiogenesis.Methods and Results:Forty human aortic segments, each harvested from a different donor, were classified as healthy or as bearing early atheromatous lesions, including fatty streaks and fibrolipidic atheroma, according to their histological features. Immunostaining for blood vessels and vascular endothelial growth factor-A (VEGF-A), as well as measurement of VEGF-A protein and mRNA levels by ELISA and real-time PCR, revealed that angiogenesis and VEGF-A production were enhanced in the medial layer of atheromatous aortas. The intramedial vessel density and invasiveness and the production of VEGF-A by medial SMCs were indeed increased in atheromatous aortas compared with healthy aortas. Furthermore, intimal layers of atheromatous aortas were enriched in soluble lipid mediators capable of inducing a sustained increase in VEGF-A production by medial SMCs, turning these cells into potent inducers of angiogenesis when incorporated into mouse Matrigel implants. Both effects were inhibited by the peroxisome proliferator-activated receptor-γ inhibitor GW9662 and mimicked by its agonist, rosiglitazone.Conclusions:We show that VEGF-A production is upregulated in medial SMCs of human atheromatous aortas and that peroxisome proliferator-activated receptor-γ agonists derived from early intimal lesions are likely to contribute to this phenotypic change. Our findings suggest that medial SMCs are central organizers of an angiogenic response initiated by the subendothelial accumulation of atherogenic lipids.

Uncoupling of nitric oxide synthase (NOS) has been implicated in left ventricular (LV) remodeling and dysfunction after myocardial infarction (MI). We hypothesized that inducible NOS (iNOS) plays a crucial role in LV remodeling after MI depending on its coupling status. MI was created in wild-type (WT), iNOS-knockout (iNOS(-/-)), endothelial NOS-knockout (eNOS(-/-)), and neuronal NOS-knockout (nNOS(-/-)) mice. iNOS and nNOS expressions were increased after MI associated with an increase in nitrotyrosine (NT) formation. The area of myocardial fibrosis and LV end-diastolic volume and ejection fraction were more deteriorated in eNOS(-/-) mice compared to other genotypes of mice 4 weeks after MI. The expression of GTP cyclohydrolase was reduced, and tetrahydrobiopterin (BH4) was depleted in the heart after MI. Oral administration of sepiapterin after MI increased dihydrobiopterin (BH2), BH4 and BH4/BH2 ratio in the infarcted but not sham-operated heart. The increase in BH4/BH2 ratio was associated with inhibition of NT formation and an increase in nitrite plus nitrate (NOx). However, this inhibition of NOS uncoupling was blunted in iNOS(-/-) mice. L-NAME abrogated sepiapterin-induced increase in NOx and angiogenesis, and blocked the beneficial effects of sepiapterin on LV remodeling and function. These results suggest that sepiapterin enhances angiogenesis and functional recovery after MI by activating the salvage pathway for BH4 synthesis and increasing bioavailable NO predominantly derived from iNOS. Sepiapterin increased capillary density and prevented LV remodeling and dysfunction after MI in WT, eNOS(-/-) and nNOS(-/-) but not iNOS(-/-) mice. L-NAME abrogated sepiapterin-induced increase in NOx and angiogenesis, and blocked the beneficial effects of sepiapterin on LV remodeling and function. These results suggest that sepiapterin enhances angiogenesis and functional recovery after MI by activating the salvage pathway for BH4 synthesis and increasing bioavailable NO predominantly derived from iNOS.

Abstract

Background- Myocardial infarction leads to cardiac remodeling and development of heart failure. Insufficient myocardial capillary density after myocardial infarction has been identified as a critical event in this process, although the underlying mechanisms of cardiac angiogenesis are mechanistically not well understood. Methods and Results- Here, we show that the small noncoding RNA microRNA-24 (miR-24) is enriched in cardiac endothelial cells and considerably upregulated after cardiac ischemia. MiR-24 induces endothelial cell apoptosis, abolishes endothelial capillary network formation on Matrigel, and inhibits cell sprouting from endothelial spheroids. These effects are mediated through targeting of the endothelium-enriched transcription factor GATA2 and the p21-activated kinase PAK4, which were identified by bioinformatic predictions and validated by luciferase gene reporter assays. Respective downstream signaling cascades involving phosphorylated BAD (Bcl-XL/Bcl-2-associated death promoter) and Sirtuin1 were identified by transcriptome, protein arrays, and chromatin immunoprecipitation analyses. Overexpression of miR-24 or silencing of its targets significantly impaired angiogenesis in zebrafish embryos. Blocking of endothelial miR-24 limited myocardial infarct size of mice via prevention of endothelial apoptosis and enhancement of vascularity, which led to preserved cardiac function and survival. Conclusions- Our findings indicate that miR-24 acts as a critical regulator of endothelial cell apoptosis and angiogenesis and is suitable for therapeutic intervention in the setting of ischemic heart disease