Shusheng Wang

The Endothelial-Specific MicroRNA miR-126 Governs Vascular Integrity and Angiogenesis

Endothelial cells play essential roles in maintenance of vascular integrity, angiogenesis, and wound repair. We show that an endothelial cell-restricted microRNA (miR-126) mediates developmental angiogenesis in vivo. Targeted deletion of miR-126 in mice causes leaky vessels, hemorrhaging, and partial embryonic lethality, due to a loss of vascular integrity and defects in endothelial cell proliferation, migration, and angiogenesis. The subset of mutant animals that survives displays defective cardiac neovascularization following myocardial infarction. The vascular abnormalities of miR-126 mutant mice resemble the consequences of diminished signaling by angiogenic growth factors, such as VEGF and FGF. Accordingly, miR-126 enhances the proangiogenic actions of VEGF and FGF and promotes blood vessel formation by repressing the expression of Spred-1, an intracellular inhibitor of angiogenic signaling. These findings have important therapeutic implications for a variety of disorders involving abnormal angiogenesis and vascular leakage.

Delivery of MicroRNA-126 by Apoptotic Bodies Induces CXCL12-Dependent Vascular Protection

Apoptotic endothelial cells release microRNA-containing microvesicles to modulate the responses of neighboring cells and reduce atherosclerosis in mice. Sounding the Alarm In addition to its importance in development and homeostasis, apoptotic cell death is implicated in a number of diseases, including atherosclerosis. Apoptotic endothelial cells at atherosclerotic plaques release microvesicles known as apoptotic bodies into the circulation, and their abundance correlates with negative indicators of disease. Zernecke et al. showed that apoptotic bodies from endothelial cells contained microRNA-126 (miR-126). Neighboring vascular cells took up the microvesicles, which allowed miR-126 to reduce the abundance of an inhibitor of the signaling of the chemokine receptor CXCR4, resulting in the increased production of CXCL12, the ligand for CXCR4. CXCL12 mediated the recruitment to atherosclerotic plaques of progenitor cells from the bone marrow, which limited plaque size. Apoptotic bodies isolated from human patients with atherosclerosis reduced the size of plaques in different mouse models of atherosclerosis. Thus, dying endothelial cells send alarm signals in the form of packaged microRNA to neighboring cells to trigger a healing response that reduces atherosclerosis. Apoptosis is a pivotal process in embryogenesis and postnatal cell homeostasis and involves the shedding of membranous microvesicles termed apoptotic bodies. In response to tissue damage, the CXC chemokine CXCL12 and its receptor CXCR4 counteract apoptosis and recruit progenitor cells. Here, we show that endothelial cell–derived apoptotic bodies are generated during atherosclerosis and convey paracrine alarm signals to recipient vascular cells that trigger the production of CXCL12. CXCL12 production was mediated by microRNA-126 (miR-126), which was enriched in apoptotic bodies and repressed the function of regulator of G protein (heterotrimeric guanosine triphosphate–binding protein) signaling 16, an inhibitor of G protein–coupled receptor (GPCR) signaling. This enabled CXCR4, a GPCR, to trigger an autoregulatory feedback loop that increased the production of CXCL12. Administration of apoptotic bodies or miR-126 limited atherosclerosis, promoted the incorporation of Sca-1+ progenitor cells, and conferred features of plaque stability on different mouse models of atherosclerosis. This study highlights functions of microRNAs in health and disease that may extend to the recruitment of progenitor cells during other forms of tissue repair or homeostasis.

AngiomiRs—Key Regulators of Angiogenesis

The formation of new blood vessels through the process of angiogenesis is critical in vascular development and homeostasis. Aberrant angiogenesis leads to a variety of diseases, such as ischemia and cancer. Recent studies have revealed important roles for miRNAs in regulating endothelial cell (EC) function, especially angiogenesis. Mice with EC-specific deletion of Dicer, a key enzyme for generating miRNAs, display defective postnatal angiogenesis. Specific miRNAs (angiomiRs) have recently been shown to regulate angiogenesis in vivo. miRNA-126, an EC-restricted miRNA, regulates vascular integrity and developmental angiogenesis. miR-378, miR-296, and the miR-17-92 cluster contribute to tumor angiogenesis. Manipulating angiomiRs in the settings of pathological vascularization represents a new therapeutic approach.

Regulation of angiogenesis and choroidal neovascularization by members of microRNA-23∼27∼24 clusters

MicroRNAs (miRNAs) modulate complex physiological and pathological processes by repressing expression of multiple components of cellular regulatory networks. Here we demonstrate that miRNAs encoded by the miR-23∼27∼24 gene clusters are enriched in endothelial cells and highly vascularized tissues. Inhibition of miR-23 and miR-27 function by locked nucleic acid-modified anti-miRNAs represses angiogenesis in vitro and postnatal retinal vascular development in vivo. Moreover, miR-23 and miR-27 are required for pathological angiogenesis in a laser-induced choroidal neovascularization mouse model. MiR-23 and miR-27 enhance angiogenesis by promoting angiogenic signaling through targeting Sprouty2 and Sema6A proteins, which exert antiangiogenic activity. Manipulating miR-23/27 levels may have important therapeutic implications in neovascular age-related macular degeneration and other vascular disorders.

Control of endothelial cell proliferation and migration by VEGF signaling to histone deacetylase 7

VEGF has been shown to regulate endothelial cell (EC) proliferation and migration. However, the nuclear mediators of the actions of VEGF in ECs have not been fully defined. We show that VEGF induces the phosphorylation of three conserved serine residues in histone deacetylase 7 (HDAC7) via protein kinase D, which promotes nuclear export of HDAC7 and activation of VEGF-responsive genes in ECs. Expression of a signal-resistant HDAC7 mutant protein in ECs inhibits proliferation and migration in response to VEGF. These results demonstrate that phosphorylation of HDAC7 serves as a molecular switch to mediate VEGF signaling and endothelial function.

MicroRNA-218 regulates vascular patterning by modulation of Slit-Robo signaling

Rationale: Establishment of a functional vasculature requires the interconnection and remodeling of nascent blood vessels. Precise regulation of factors that influence endothelial cell migration and function is essential for these stereotypical vascular patterning events. The secreted Slit ligands and their Robo receptors constitute a critical signaling pathway controlling the directed migration of both neurons and vascular endothelial cells during embryonic development, but the mechanisms of their regulation are incompletely understood. Objective: To identify microRNAs regulating aspects of the Slit-Robo pathway and vascular patterning. Methods and Results: Here, we provide evidence that microRNA (miR)-218, which is encoded by an intron of the Slit genes, inhibits the expression of Robo1 and Robo2 and multiple components of the heparan sulfate biosynthetic pathway. Using in vitro and in vivo approaches, we demonstrate that miR-218 directly represses the expression of Robo1, Robo2, and glucuronyl C5-epimerase (GLCE), and that an intact miR-218–Slit–Robo regulatory network is essential for normal vascularization of the retina. Knockdown of miR-218 results in aberrant regulation of this signaling axis, abnormal endothelial cell migration, and reduced complexity of the retinal vasculature. Conclusions: Our findings link Slit gene expression to the posttranscriptional regulation of Robo receptors and heparan sulfate biosynthetic enzymes, allowing for precise control over vascular guidance cues influencing the organization of blood vessels during development.

miRNAs as potential therapeutic targets for age-related macular degeneration

Since their recent discovery, miRNAs have been shown to play critical roles in a variety of pathophysiological processes. Such processes include pathological angiogenesis, the oxidative stress response, immune response and inflammation, all of which have been shown to have important and interdependent roles in the pathogenesis and progression of age-related macular degeneration (AMD). Here we present a brief review of the pathological processes involved in AMD and review miRNAs and other noncoding RNAs involved in regulating these processes. Specifically, we discuss several candidate miRNAs that show promise as AMD therapeutic targets due to their direct involvement in choroidal neovascularization or retinal pigment epithelium atrophy. We discuss potential miRNA-based therapeutics and delivery methods for AMD and provide future directions for the field of miRNA research with respect to AMD. We believe the future of miRNAs in AMD therapy is promising.

Keep your eyes open: challenges and opportunities in Ophthalmic Therapeutics

The eyes are our windows to the world. Major ocular diseases, including cataracts, glaucoma, age-related macular degeneration (AMD), diabetic retinopathy (DR), dry eye syndrome and allergic conjunctivitis, can cause vision impairment and even blindness, which not only affect our daily life, but also have a significant socio-economic and psychological impact on our society. The prevalence of vision impairment was estimated to be 147 million people in 2010, and is projected to reach 191 million in the word in 2020 due to an increase in aging populations. The market for ophthalmic disease therapeutics is expected to reach

Primitive Erythropoiesis Is Regulated by miR-126 via Nonhematopoietic Vcam-1+ Cells

18.7 billion this year, and is predicted to grow steadily. Ophthalmic drug development has been in the forefront of therapeutic research partly because the eye is an accessible and immunologically isolated organ, which has the advantage of avoiding the complications of systemic drug delivery. With the recent advances in genetics, genomics and stem cell biology, the mechanisms of many ophthalmic diseases are being increasingly revealed. Accordingly, the field of ophthalmic therapeutics is facing unprecedented challenges and opportunities. Looking back in history, ophthalmic drug development has been driven by the scientific breakthroughs. For example, the discovery that vascular endothelial growth factor (VEGF) is a driving force of choroidal neovascularization in AMD has led to the successful development of anti-VEGF antibodies for wet AMD therapy. The discovery that prostaglandin mediates reduction in intraocular pressure (IOP) has led to the treatment of ocular hypertension in glaucoma using prostaglandin analogues. Research over the past decade has uncovered novel mechanisms for many ocular diseases, which will likely spawn new therapeutic solutions. But this field is not without challenges: (A) currently there is no cure for many ocular diseases, including dry AMD, DR and glaucoma; (B) current ophthalmic drug development is not based on the unique characteristics of ocular diseases, but mainly applies agents developed for non-ocular diseases to the eye; (C) most of the ocular diseases are multi-factorial diseases with both genetic and environmental (nutritional) risk factors, therefore many drugs only work for a subset of patients. For example, cyclosporine, acting as an immune-modulator for dry-eye disease, is only effective in about 15% of all patients; (D) ophthalmic drug delivery, especially to the posterior eye, remains a considerable challenge. These challenges present unique opportunities for researchers in the field of ophthalmology to further elucidate the mechanisms of and develop innovative therapeutics and delivery approaches for ocular diseases. Despite the challenges, significant progress has recently been made towards understanding the mechanisms of several ocular diseases, which may translate into future therapeutics. Dry AMD accounts for up to 90% of the AMD cases and is currently without cure. Polymorphisms in members of the complement system, especially complement factor H (CFH), have been associated with AMD. Moreover, CFH was recently shown to protect against oxidative stress-induced inflammation in animal model. Inhibitors of the complement system and the recombinant form of CFH are being tested preclinically for AMD treatment. Aβ amyloid, originally implicated in Alzheimer’s disease, was recently associated with pathogenesis of AMD. Anti-Aβ amyloid antibody has shown promise in animal models of AMD. Autophagy is currently being investigated for AMD involvement, and may represent a potential therapeutics for AMD (See review in this issue). Diabetic retinopathy is one of the leading causes of blindness in the working class. Besides laser photocoagulation and vitrectomy surgery, several therapeutic options, including protein kinase inhibitors, cyclooxygenase inhibitors, anti-VEGF and slow release steroid, are on clinical trials. Some of them may enter the market in the near future. As reviewed in this issue, pericytes and the angiopoietin (Ang)-Tie-2 signaling also play an important role in the development of DR, and may be potential therapeutic targets for DR. For glaucoma, the current mainstay for treatment is to lower IOP, therefore preventing further damage to the optic nerve. β-blocker, prostaglandin, carbonic anhydrase inhibitor, miotics, α-adrenergic agonist, or their combinations are routinely used for lowering IOP. Rho kinase inhibitors and actin depolymerization agents are currently tested in clinical trials for safely reducing IOP. Dominant mutations in the olfactomedin (OLF) domain of myocilin have been associated with several populations of familial glaucoma, and molecular studies suggest that the OLF domain of myocilin may be a bona fide target for future glaucoma therapeutics (reviewed in this issue). Looking into future, the explosion in basic research in ocular biology and disease will lead to revolutionarily innovative ophthalmic therapeutics. Numerous opportunities also present themselves to researchers working on medicinal chemistry, new biomaterials, and new delivery system (see reviews for these subjects in the issue). With the coordinated effort of all these groups, the future of ophthalmic therapeutics is bright.