Surface Electrical Impedance Myography Detects Skeletal Muscle Atrophy in Aged Wildtype Zebrafish and Aged gpr27 Knockout Zebrafish.

Throughout a vertebrate organism's lifespan, skeletal muscle mass and function progressively decline. This age-related condition is termed sarcopenia. In humans, sarcopenia is associated with risk of falling, cardiovascular disease, and all-cause mortality. As the world population ages, projected to reach 2 billion older adults worldwide in 2050, the economic burden on the healthcare system is also projected to increase considerably. Currently, there are no pharmacological treatments for sarcopenia, and given the long-term nature of aging studies, high-throughput chemical screens are impractical in mammalian models. Zebrafish is a promising, up-and-coming vertebrate model in the field of sarcopenia that could fill this gap. Here, we developed a surface electrical impedance myography (sEIM) platform to assess skeletal muscle health, quantitatively and noninvasively, in adult zebrafish (young, aged, and genetic mutant animals). In aged zebrafish (~85% lifespan) as compared to young zebrafish (~20% lifespan), sEIM parameters (2 kHz phase angle, 2 kHz reactance, and 2 kHz resistance) robustly detected muscle atrophy (p < 0.000001, q = 0.000002; p = 0.000004, q = 0.000006; p = 0.000867, q = 0.000683, respectively). Moreover, these same measurements exhibited strong correlations with an established morphometric parameter of muscle atrophy (myofiber cross-sectional area), as determined by histological-based morphometric analysis (r = 0.831, p = 2 × 10-12; r = 0.6959, p = 2 × 10-8; and r = 0.7220; p = 4 × 10-9, respectively). Finally, the genetic deletion of gpr27, an orphan G-protein coupled receptor (GPCR), exacerbated the atrophy of skeletal muscle in aged animals, as evidenced by both sEIM and histology. In conclusion, the data here show that surface EIM techniques can effectively discriminate between healthy young and sarcopenic aged muscle as well as the advanced atrophied muscle in the gpr27 KO animals. Moreover, these studies show how EIM values correlate with cell size across the animals, making it potentially possible to utilize sEIM as a "virtual biopsy" in zebrafish to noninvasively assess myofiber atrophy, a valuable measure for muscle and gerontology research.

Predicting myofiber cross-sectional area and triglyceride content with electrical impedance myography: A study in db/db mice.

BACKGROUND: Electrical impedance myography (EIM) provides insight into muscle composition and structure. We sought to evaluate its use in a mouse obesity model characterized by myofiber atrophy. METHODS: We applied a prediction algorithm, ie, the least absolute shrinkage and selection operator (LASSO), to surface, needle array, and ex vivo EIM data from db/db and wild-type mice and assessed myofiber cross-sectional area (CSA) histologically and triglyceride (TG) content biochemically. RESULTS: EIM data from all three modalities provided acceptable predictions of myofiber CSA with average root mean square error (RMSE) of 15% in CSA (ie, ±209 µm2 for a mean CSA of 1439 µm2 ) and TG content with RMSE of 30% in TG content (ie, ±7.3 nmol TG/mg muscle for a mean TG content of 25.4 nmol TG/mg muscle). CONCLUSIONS: EIM combined with a predictive algorithm provides reasonable estimates of myofiber CSA and TG content without the need for biopsy.

Electrical impedance myography for the detection of muscle inflammation induced by λ-carrageenan.

Electrical impedance myography (EIM) is a technique for the assessment of muscle health and composition and has been shown to be sensitive to a variety of muscle pathologies including neurogenic atrophy and connective tissue deposition. However, it has been minimally studied in pure inflammation. In this study, we sought to assess EIM sensitivity to experimental inflammation induced by the localized intramuscular injection of λ-carrageenan. A total of 91 mice underwent 1-1000 kHz EIM measurements of gastrocnemius using a needle array, followed by injection of either 0.3% λ-carrageenan in phosphate-buffered saline (PBS) or PBS alone. Animals were then remeasured with EIM at 4, 24, 48, or 72 hours and euthanized and quantitative assessment of muscle histology was performed. Parallel alterations in both 5 and 50 kHz EIM values were identified at 4 and 24 hours, including reductions in phase, reactance, and resistance. In PBS-treated animals these values normalized by 48 hours, whereas substantial reductions in phase and reactance in 5 kHz EIM values persisted at 48 and 72 hours (i.e., values of phase 72 hours post-injection were 6.51 ± 0.40 degrees for λ-carrageenan versus 8.44 ± 0.35 degrees for PBS p<0.001, n = 11 per group). The degree of basophilic area observed in muscle sections by histology correlated to the degree of phase change at these two time points (Rspearman = -0.51, p = 0.0029). Changes in low frequency EIM parameters are sensitive to the presence of inflammatory infiltrates, and have the potential of serving as a simple means of quantifying the presence and extent of muscle inflammation without the need for biopsy.

Estimating Myofiber Size With Electrical Impedance Myography: a Study In Amyotrophic Lateral Sclerosis MICE.

INTRODUCTION: A method for quantifying myofiber size noninvasively would find wide use, including primary diagnosis and evaluating response to therapy. METHODS: Using prediction algorithms, including the least absolute shrinkage and selection operator, we applied multifrequency electrical impedance myography (EIM) to amyotrophic lateral sclerosis superoxide dismutase 1 G93A mice of different ages and assessed myofiber size histologically. RESULTS: Multifrequency EIM data provided highly accurate predictions of myofiber size, with a root mean squared error (RMSE) of only 14% in mean myofiber area (corresponding to ± 207 µm2 for a mean area of 1,488 µm2 ) and an RMSE of only 8.8% in predicting the coefficient of variation in fiber size distribution. DISCUSSION: This impedance-based approach provides predictive variables to assess myofiber size and distribution with good accuracy, particularly in diseases in which myofiber atrophy is the predominant histological feature, without the requirement for biopsy or burdensome quantification. Muscle Nerve 58: 713-717, 2018.

Early Actions of Anti-Vascular Endothelial Growth Factor/Vascular Endothelial Growth Factor Receptor Drugs on Angiogenic Blood Vessels.

Tumors induce their heterogeneous vasculature by secreting vascular endothelial growth factor (VEGF)-A. Anti-VEGF/VEGF receptor (VEGFR) drugs treat cancer, but the underlying mechanisms remain unclear. An adenovirus expressing VEGF-A (Ad-VEGF-A164) replicates the tumor vasculature in mice without tumor cells. Mother vessels (MV) are the first angiogenic vessel type to form in tumors and after Ad-VEGF-A164. Multiday treatments with a VEGF trap reverted MV back to normal microvessels. We now show that, within hours, a single dose of several anti-VEGF drugs collapsed MV to form glomeruloid microvascular proliferations (GMP), accompanied by only modest endothelial cell death. GMP, common in many human cancers but of uncertain origin, served as an intermediary step in MV reversion to normal microvessels. The vasodisruptive drug combretastatin CA4 also targeted MV selectively but acted differently, extensively killing MV endothelium. Antivascular changes were quantified with a novel Evans blue dye assay that measured vascular volumes. As in tumors, Ad-VEGF-A164 strikingly increased endothelial nitric oxide synthase (eNOS) expression. The eNOS inhibitor N(G)-Nitro-l-arginine methyl ester mimicked anti-VEGF/VEGFR drugs, rapidly collapsing MV to GMP. Inhibition of eNOS reduces synthesis of its vasodilatory product, nitric oxide, leading to arterial contraction. Patients and mice receiving anti-VEGF/VEGFR drugs develop hypertension, reflecting systemic arterial contraction. Together, anti-VEGF/VEGFR drugs act in part by inhibiting eNOS, causing vasocontraction, MV collapse to GMP, and subsequent reversion of GMP to normal microvessels, all without extensive vascular killing.

Stromal-Based Signatures for the Classification of Gastric Cancer.

Treatment of metastatic gastric cancer typically involves chemotherapy and monoclonal antibodies targeting HER2 (ERBB2) and VEGFR2 (KDR). However, reliable methods to identify patients who would benefit most from a combination of treatment modalities targeting the tumor stroma, including new immunotherapy approaches, are still lacking. Therefore, we integrated a mouse model of stromal activation and gastric cancer genomic information to identify gene expression signatures that may inform treatment strategies. We generated a mouse model in which VEGF-A is expressed via adenovirus, enabling a stromal response marked by immune infiltration and angiogenesis at the injection site, and identified distinct stromal gene expression signatures. With these data, we designed multiplexed IHC assays that were applied to human primary gastric tumors and classified each tumor to a dominant stromal phenotype representative of the vascular and immune diversity found in gastric cancer. We also refined the stromal gene signatures and explored their relation to the dominant patient phenotypes identified by recent large-scale studies of gastric cancer genomics (The Cancer Genome Atlas and Asian Cancer Research Group), revealing four distinct stromal phenotypes. Collectively, these findings suggest that a genomics-based systems approach focused on the tumor stroma can be used to discover putative predictive biomarkers of treatment response, especially to antiangiogenesis agents and immunotherapy, thus offering an opportunity to improve patient stratification. Cancer Res; 76(9); 2573-86. ©2016 AACR.

PGC-1α induces SPP1 to activate macrophages and orchestrate functional angiogenesis in skeletal muscle.

RATIONALE: Mechanisms of angiogenesis in skeletal muscle remain poorly understood. Efforts to induce physiological angiogenesis hold promise for the treatment of diabetic microvascular disease and peripheral artery disease but are hindered by the complexity of physiological angiogenesis and by the poor angiogenic response of aged and patients with diabetes mellitus. To date, the best therapy for diabetic vascular disease remains exercise, often a challenging option for patients with leg pain. Peroxisome proliferation activator receptor-γ coactivator-1α (PGC-1α), a powerful regulator of metabolism, mediates exercise-induced angiogenesis in skeletal muscle. OBJECTIVE: To test whether, and how, PGC-1α can induce functional angiogenesis in adult skeletal muscle. METHODS AND RESULTS: Here, we show that muscle PGC-1α robustly induces functional angiogenesis in adult, aged, and diabetic mice. The process involves the orchestration of numerous cell types and leads to patent, nonleaky, properly organized, and functional nascent vessels. These findings contrast sharply with the disorganized vasculature elicited by induction of vascular endothelial growth factor alone. Bioinformatic analyses revealed that PGC-1α induces the secretion of secreted phosphoprotein 1 and the recruitment of macrophages. Secreted phosphoprotein 1 stimulates macrophages to secrete monocyte chemoattractant protein-1, which then activates adjacent endothelial cells, pericytes, and smooth muscle cells. In contrast, induction of PGC-1α in secreted phosphoprotein 1(-/-) mice leads to immature capillarization and blunted arteriolarization. Finally, adenoviral delivery of PGC-1α into skeletal muscle of either young or old and diabetic mice improved the recovery of blood flow in the murine hindlimb ischemia model of peripheral artery disease. CONCLUSIONS: PGC-1α drives functional angiogenesis in skeletal muscle and likely recapitulates the complex physiological angiogenesis elicited by exercise.

RhoB controls coordination of adult angiogenesis and lymphangiogenesis following injury by regulating VEZF1-mediated transcription.

Mechanisms governing the distinct temporal dynamics that characterize post-natal angiogenesis and lymphangiogenesis elicited by cutaneous wounds and inflammation remain unclear. RhoB, a stress-induced small GTPase, modulates cellular responses to growth factors, genotoxic stress and neoplastic transformation. Here we show, using RhoB null mice, that loss of RhoB decreases pathological angiogenesis in the ischaemic retina and reduces angiogenesis in response to cutaneous wounding, but enhances lymphangiogenesis following both dermal wounding and inflammatory challenge. We link these unique and opposing roles of RhoB in blood versus lymphatic vasculatures to the RhoB-mediated differential regulation of sprouting and proliferation in primary human blood versus lymphatic endothelial cells. We demonstrate that nuclear RhoB-GTP controls expression of distinct gene sets in each endothelial lineage by regulating VEZF1-mediated transcription. Finally, we identify a small-molecule inhibitor of VEZF1-DNA interaction that recapitulates RhoB loss in ischaemic retinopathy. Our findings establish the first intra-endothelial molecular pathway governing the phased response of angiogenesis and lymphangiogenesis following injury.

Heterogeneity of the tumor vasculature: the need for new tumor blood vessel type-specific targets.

Therapies directed against VEGF-A and its receptors are effective in treating many mouse tumors but have been less so in treating human cancer patients. To elucidate the reasons that might be responsible for this difference in response, we investigated the nature of the blood vessels that appear in human and mouse cancers and the tumor "surrogate" blood vessels that develop in immunodeficient mice in response to an adenovirus expressing VEGF-A(164). Both tumor and tumor surrogate blood vessels are heterogeneous and form by two distinct processes, angiogenesis and arterio-venogenesis. The first new angiogenic blood vessels to form are mother vessels (MV); MV arise from preexisting venules and capillaries and evolve over time into glomeruloid microvascular proliferations (GMP) and subsequently into capillaries and vascular malformations (VM). Arterio-venogenesis results from the remodeling and enlargement of preexisting arteries and veins, leading to the formation of feeder arteries (FA) and draining veins (DV) that supply and drain angiogenic vessels. Of these different blood vessel types, only the two that form first, MV and GMP, were highly responsive to anti-VEGF therapy, whereas "late"-formed capillaries, VM, FA and DV were relatively unresponsive. This finding may explain, at least in part, the relatively poor response of human cancers to anti-VEGF/VEGFR therapies, because human cancers, present for months or years prior to discovery, are expected to contain a large proportion of late-formed blood vessels. The future of anti-vascular cancer therapy may depend on finding new targets on "late" vessels, apart from those associated with the VEGF/VEGFR axis.

Anti-VEGF/VEGFR therapy for cancer: reassessing the target.

Judah Folkman recognized that new blood vessel formation is important for tumor growth and proposed antiangiogenesis as a novel approach to cancer therapy. Discovery of vascular permeability factor VEGF-A as the primary tumor angiogenesis factor prompted the development of a number of drugs that targeted it or its receptors. These agents have often been successful in halting tumor angiogenesis and in regressing rapidly growing mouse tumors. However, results in human cancer have been less impressive. A number of reasons have been offered for the lack of greater success, and, here, we call attention to the heterogeneity of the tumor vasculature as an important issue. Human and mouse tumors are supplied by at least 6 well-defined blood vessel types that arise by both angiogenesis and arterio-venogenesis. All 6 types can be generated in mouse tissues by an adenoviral vector expressing VEGF-A(164). Once formed, 4 of the 6 types lose their VEGF-A dependency, and so their responsiveness to anti-VEGF/VEGF receptor therapy. If therapies directed against the vasculature are to have a greater impact on human cancer, targets other than VEGF and its receptors will need to be identified on these resistant tumor vessels.

Vascular hyperpermeability, angiogenesis, and stroma generation.

It has been known for more than half a century that the tumor microvasculature is hyperpermeable to plasma proteins. However, the identity of the leaky vessels and the consequences of vascular hyperpermeability have received little attention. This article places tumor vascular hyperpermeability in a broader context, relating it to (1) the low-level "basal" permeability of the normal vasculature; (2) the "acute," short-term hyperpermeability induced by vascular permeability factor/vascular endothelial growth factor (VPF/VEGF-A) and other vascular permeabilizing agents; and (3) the "chronic" hyperpermeability associated with longer-term exposure to agents such as VPF/VEGF-A that accompanies many types of pathological angiogenesis. Leakage of plasma protein-rich fluids is important because it activates the clotting system, depositing an extravascular fibrin gel provisional matrix that serves as the first step in stroma generation.

Tumor-surrogate blood vessel subtypes exhibit differential susceptibility to anti-VEGF therapy.

Antivascular therapy directed against VEGF or its receptors (VEGFR) has been successful when administered at early stages of tumor vessel growth but is less effective when administered later. Tumor blood vessels are heterogeneous, so vessel subpopulations may differ in their requirements for tumor cell-secreted VEGF and in their susceptibility to anti-VEGF/VEGFR therapy. Human cancers contain several distinct blood vessel types, including mother vessels (MV), glomeruloid microvascular proliferations (GMP), vascular malformations (VM), feeding arteries (FA), and draining veins (DV), all of which can be generated in mice in the absence of tumor cells using expression vectors for VEGF-A(164). In this study, we investigated the sensitivity of each of these vessel types to anti-VEGF therapy with Aflibercept (VEGF Trap), a potent inhibitor of VEGF-A(164). Administering VEGF Trap treatment before or shortly after injection of a recombinant VEGF-A(164)-expressing adenovirus could prevent or regress tumor-free neovasculature, but it was progressively less effective if initiated at later times. Early-forming MVs and GMPs in which the lining endothelial cells expressed high levels of VEGFR-2 were highly susceptible to blockade by VEGF Trap. In contrast, late-forming VMs, FAs, and DVs that expressed low levels of VEGFR-2 were largely resistant. Together, our findings define the susceptibility of different blood vessel subtypes to anti-VEGF therapy, offering a possible explanation for the limited effectiveness of anti-VEGF-A/VEGFR treatment of human cancers, which are typically present for months to years before discovery and are largely populated by late-forming blood vessels.

RhoJ is an endothelial cell-restricted Rho GTPase that mediates vascular morphogenesis and is regulated by the transcription factor ERG.

ERG is a member of the ETS transcription factor family that is highly enriched in endothelial cells (ECs). To further define the role of ERG in regulating EC function, we evaluated the effect of ERG knock-down on EC lumen formation in 3D collagen matrices. Blockade of ERG using siRNA completely interferes with EC lumen formation. Quantitative PCR (QPCR) was used to identify potential downstream gene targets of ERG. In particular, we identified RhoJ as the Rho GTPase family member that is closely related to Cdc42 as a target of ERG. Knockdown of ERG expression in ECs led to a 75% reduction in the expression of RhoJ. Chromatin immunoprecipitation and transactivation studies demonstrated that ERG could bind to functional sites in the proximal promoter of the RhoJ gene. Knock-down of RhoJ similarly resulted in a marked reduction in the ability of ECs to form lumens. Suppression of either ERG or RhoJ during EC lumen formation was associated with a marked increase in RhoA activation and a decrease in Rac1 and Cdc42 activation and their downstream effectors. Finally, in contrast to other Rho GTPases, RhoJ exhibits a highly EC-restricted expression pattern in several different tissues, including the brain, heart, lung, and liver.

Proteolytic cleavage of versican and involvement of ADAMTS-1 in VEGF-A/VPF-induced pathological angiogenesis.

Malignant tumors and chronic inflammatory diseases induce angiogenesis by overexpressing vascular endothelial growth factor A (VEGF-A/VPF). VEGF-A-induced pathological angiogenesis can be mimicked in immunoincompetent mice with an adenoviral vector expressing VEGF-A(164) (Ad-VEGF-A(164)). The initial step is generation of greatly enlarged "mother" vessels (MV) from preexisting normal venules by a process involving degradation of their rigid basement membranes. Immunohistochemical and Western blot analyses revealed that versican, an extracellular matrix component in the basement membranes of venules, is degraded early in the course of MV formation, resulting in the appearance of a versican N-terminal DPEAAE fragment associated with MV endothelial cells. The protease ADAMTS-1, known to cleave versican near its N terminus to generate DPEAAE, is also upregulated by VEGF-A in parallel with MV formation and localizes to the endothelium of the developing MV. The authors also show that MMP-15 (MT-2 MMP), a protease that activates ADAMTS-1, is upregulated by VEGF-A in endothelial cells in vitro and in vivo. These data suggest VEGF-A initiates MV formation, in part, by inducing the expression of endothelial cell proteases such as ADAMTS-1 and MMP-15 that act in concert to degrade venular basement membrane versican. Thus, versican is actively processed during the early course of VEGF-A-induced pathological angiogenesis.

Cdc42-mediated inhibition of GSK-3ß improves angio-architecture and lumen formation during VEGF-driven pathological angiogenesis.

Vascular endothelial growth factor-A (VEGF) typically induces abnormal angiogenesis in the adult, thereby aggravating disease pathology and limiting utility of VEGF for therapeutic angiogenesis. To identify strategies for rectifying defects in pathological VEGF neovessels, we investigated consequences of modulating the Rho GTPase Cdc42. In a mouse skin model of VEGF-driven pathological angiogenesis, transduction with active Cdc42 (L28Cdc42) markedly improved VEGF neovessels, as measured by increased lumen formation, enlarged vessel diameter, and enhanced perfusion of macromolecular tracers. Conversely, transduction with dominant negative Cdc42 (N17Cdc42) impaired endothelial cell (EC) assembly into lumenized blood vessels and reduced neovessel diameter and tracer perfusion. In vitro, active Cdc42 improved coordination between actin filaments and microtubules and enhanced formation of vascular cords, suggesting that active Cdc42 rectifies defects in angiogenesis by improving cytoskeletal dynamics and capillary morphogenesis. Analyses of Cdc42 signaling in microvascular ECs indicated that active Cdc42 also inhibits glycogen synthase kinase-3ß (GSK-3ß), a multi-functional serine/threonine protein kinase. Pharmacological inhibition of GSK-3ß improved vascular cord formation in vitro and promoted proper neovessel formation in vivo comparably to active Cdc42, thus linking GSK-3ß inhibition to the mechanism by which active Cdc42 rectifies pathological neovascularization. These studies identify activation of Cdc42 and inhibition of GSK-3ß as novel strategies for correcting abnormalities associated with VEGF-driven angiogenesis, and they suggest new approaches for achieving improved therapeutic neovascularization with VEGF.

Moderation of calpain activity promotes neovascular integration and lumen formation during VEGF-induced pathological angiogenesis.

BACKGROUND: Successful neovascularization requires that sprouting endothelial cells (ECs) integrate to form new vascular networks. However, architecturally defective, poorly integrated vessels with blind ends are typical of pathological angiogenesis induced by vascular endothelial growth factor-A (VEGF), thereby limiting the utility of VEGF for therapeutic angiogenesis and aggravating ischemia-related pathologies. Here we investigated the possibility that over-exuberant calpain activity is responsible for aberrant VEGF neovessel architecture and integration. Calpains are a family of intracellular calcium-dependent, non-lysosomal cysteine proteases that regulate cellular functions through proteolysis of numerous substrates. METHODOLOGY/PRINCIPAL FINDINGS: In a mouse skin model of VEGF-driven angiogenesis, retroviral transduction with dominant-negative (DN) calpain-I promoted neovessel integration and lumen formation, reduced blind ends, and improved vascular perfusion. Moderate doses of calpain inhibitor-I improved VEGF-driven angiogenesis similarly to DN calpain-I. Conversely, retroviral transduction with wild-type (WT) calpain-I abolished neovessel integration and lumen formation. In vitro, moderate suppression of calpain activity with DN calpain-I or calpain inhibitor-I increased the microtubule-stabilizing protein tau in endothelial cells (ECs), increased the average length of microtubules, increased actin cable length, and increased the interconnectivity of vascular cords. Conversely, WT calpain-I diminished tau, collapsed microtubules, disrupted actin cables, and inhibited integration of cord networks. Consistent with the critical importance of microtubules for vascular network integration, the microtubule-stabilizing agent taxol supported vascular cord integration whereas microtubule dissolution with nocodazole collapsed cord networks. CONCLUSIONS/SIGNIFICANCE: These findings implicate VEGF-induction of calpain activity and impairment of cytoskeletal dynamics in the failure of VEGF-induced neovessels to form and integrate properly. Accordingly, calpain represents an important target for rectifying key vascular defects associated with pathological angiogenesis and for improving therapeutic angiogenesis with VEGF.

Heterogeneity of the tumor vasculature.

The blood vessels supplying tumors are strikingly heterogeneous and differ from their normal counterparts with respect to organization, structure, and function. Six distinctly different tumor vessel types have been identified, and much has been learned about the steps and mechanisms by which they form. Four of the six vessel types (mother vessels, capillaries, glomeruloid microvascular proliferations, and vascular malformations) develop from preexisting normal venules and capillaries by angiogenesis. The two remaining vessel types (feeder arteries and draining veins) develop from arterio-venogenesis, a parallel, poorly understood process that involves the remodeling of preexisting arteries and veins. All six of these tumor vessel types can be induced to form sequentially in normal mouse tissues by an adenoviral vector expressing vascular endothelial growth factor (VEGF)-A164. Current antiangiogenic cancer therapies directed at VEGF-A or its receptors have been of only limited benefit to cancer patients, perhaps because they target only the endothelial cells of the tumor blood vessel subset that requires exogenous VEGF-A for maintenance. A goal of future work is to identify therapeutic targets on tumor blood vessel endothelial cells that have lost this requirement.

Vascular permeability and pathological angiogenesis in caveolin-1-null mice.

Caveolin-1, the signature protein of endothelial cell caveolae, has many important functions in vascular cells. Caveolae are thought to be the transcellular pathway by which plasma proteins cross normal capillary endothelium, but, unexpectedly, cav-1(-/-) mice, which lack caveolae, have increased permeability to plasma albumin. The acute increase in vascular permeability induced by agents such as vascular endothelial growth factor (VEGF)-A occurs through venules, not capillaries, and particularly through the vesiculo-vacuolar organelle (VVO), a unique structure composed of numerous interconnecting vesicles and vacuoles that together span the venular endothelium from lumen to ablumen. Furthermore, the hyperpermeable blood vessels found in pathological angiogenesis, mother vessels, are derived from venules. The present experiments made use of cav-1(-/-) mice to investigate the relationship between caveolae and VVOs and the roles of caveolin-1 in VVO structure in the acute vascular hyperpermeability induced by VEGF-A and in pathological angiogenesis and associated chronic vascular hyperpermeability. We found that VVOs expressed caveolin-1 variably but, in contrast to caveolae, were present in normal numbers and with apparently unaltered structure in cav-1(-/-) mice. Nonetheless, VEGF-A-induced hyperpermeability was strikingly reduced in cav-1(-/-) mice, as was pathological angiogenesis and associated chronic vascular hyperpermeability, whether induced by VEGF-A(164) or by a tumor. Thus, caveolin-1 is not necessary for VVO structure but may have important roles in regulating VVO function in acute vascular hyperpermeability and angiogenesis.

Thrombospondin-1 modulates vascular endothelial growth factor activity at the receptor level.

Vascular endothelial growth factor (VEGF) is a well-established stimulator of vascular permeability and angiogenesis, whereas thrombospondin-1 (TSP-1) is a potent angiogenic inhibitor. In this study, we have found that the TSP-1 receptors CD36 and beta1 integrin associate with the VEGF receptor 2 (VEGFR2). The coclustering of receptors that regulate angiogenesis may provide the endothelial cell with a platform for integration of positive and negative signals in the plane of the membrane. Thus, this complex may represent a molecular switch that regulates angiogenesis and determines endothelial cell behavior. In this context, physiological levels of TSP-1 appear to support VEGFR2 function on both the cellular and tissue level, because phosphorylation of VEGFR2 and vascular permeability in response to VEGF are decreased in TSP-1-null mice and isolated endothelial cells. A therapeutic agent based on the antiangiogenic domain of TSP-1, designated 3TSR (for three TSP-1 type 1 repeats), has significant antiangiogenic and antitumor efficacy. Systemic treatment of wild-type mice with 3TSR significantly decreased VEGF-induced permeability. Consistent with this result, VEGF-stimulated phosphorylation of VEGFR2 was also significantly decreased in lung extracts from 3TSR-treated mice. Moreover, 3TSR significantly decreased VEGF-stimulated VEGFR2 phosphorylation in human dermal microvascular endothelial cells in culture. Taken together, the results indicate that TSP-1 and 3TSR modulate the function of VEGFR2.

Rapamycin inhibition of the Akt/mTOR pathway blocks select stages of VEGF-A164-driven angiogenesis, in part by blocking S6Kinase.

OBJECTIVE: We evaluated the stages of VEGF-A(164) driven angiogenesis that are inhibited by therapeutic doses of rapamycin and the potential role of S6K1 in that response. METHODS AND RESULTS: We assessed the effects of rapamycin on the several stages of angiogensis and lymphangiogenesis induced with an adenovirus expressing VEGF-A(164) (Ad-VEGF-A(164)) in the ears of adult nude mice. Rapamycin (0.5 mg/kg/d) effectively inhibited mTOR and downstream S6K1 signaling and partially inhibited Akt signaling, likely through effects on TORC2. The earliest stages of angiogenesis, including mother vessel formation and increased vascular permeability, were strikingly inhibited by rapamycin, as was subsequent formation of daughter glomeruloid microvasular proliferations. However, later stage formation of vascular malformations and lymphangiogenesis were unaffected. Retrovirally delivered isoforms and shRNAs demonstrated that S6K1 signaling plays an important role in early VEGF-A(164)-angiogenesis. CONCLUSIONS: Rapamycin potently inhibited early and mid stages of VEGF-A(164)-driven angiogenesis, but not late-stage angiogenesis or lymphangiogenesis. Rapamycin decreased phosphorylation of both Akt and S6, suggesting that both the TORC1 and TORC2 pathways are impacted. Inhibition of S6K1 signaling downstream of mTOR is a major component of the antiangiogenesis action of rapamycin.