Melissa Petreaca

Cell and molecular mechanisms of insulin-induced angiogenesis

Angiogenesis, the development of new blood vessel from pre‐existing vessels, is a key process in the formation of the granulation tissue during wound healing. The appropriate development of new blood vessels, along with their subsequent maturation and differentiation, establishes the foundation for functional wound neovasculature. We performed studies in vivo and used a variety of cellular and molecular approaches in vitro to show that insulin stimulates angiogenesis and to elucidate the signalling mechanisms by which this protein stimulates microvessel development. Mice skin injected with insulin shows longer vessels with more branches, along with increased numbers of associated α‐smooth muscle actin‐expressing cells, suggesting the appropriate differentiation and maturation of the new vessels. We also found that insulin stimulates human microvascular endothelial cell migration and tube formation, and that these effects occur independently of VEGF/VEGFR signalling, but are dependent upon the insulin receptor itself. Downstream signalling pathways involve PI3K, Akt, sterol regulatory element‐binding protein 1 (SREBP‐1) and Rac1; inhibition of these pathways results in elimination of endothelial cell migration and tube formation and significantly decreases the development of microvessels. Our findings strongly suggest that insulin is a good candidate for the treatment of ischaemic wounds and other conditions in which blood vessel development is impaired.

An assay system for in vitro detection of permeability in human "endothelium".

The molecular mechanisms by which endothelial permeability occurs are often studied more readily in vitro, underscoring the importance of the use of systems that mimic human endothelium in vivo. We present an assay that accurately models human endothelium by use of primary human microvascular endothelial cells (hMVEC), because permeability primarily occurs at the microvascular level, and transwell filter units coated with Matrigel, extracellular matrix that mimics basal lamina, the matrix that is tightly associated with endothelium and is critical for its proper function. As a tracer molecule, we used 3-kDa dextran-FITC to detect leakage through the small gaps present in the early stages of permeability induction. The permeability-inducing agents IL-8 and VEGF were added to the lower chamber of the transwell units to mimic inflammatory conditions in vivo. After optimization, we were able to minimize basal permeability and to detect rapid changes in permeability stimulated by IL-8 and VEGF, similar to that observed in vivo. Furthermore, we have used this system to delineate the importance of the transactivation of VEGFR2 in IL-8-induced permeability and have confirmed the relevance of this signaling in vivo, suggesting that our permeability assay system adequately mimics the in vivo situation. Therefore, this system can be used to better understand the molecular mechanisms of human vascular permeability in a more in vivo-like setting and, thus, may be used to test effective therapeutics to prevent and treat diseases involving persistent permeability.

Deletion of a tumor necrosis superfamily gene in mice leads to impaired healing that mimics chronic wounds in humans

Proper healing of cutaneous wounds progresses through a series of overlapping phases. Nonhealing wounds are defective in one or more of these processes and represent a major clinical problem. A critical issue in developing treatments for chronic wounds is the paucity of animal models to study the mechanisms underlying the defects in healing. Here we show that deletion of tumor necrosis factor superfamily member 14 (TNFSF14/LIGHT) leads to impaired wounds in mice that have the characteristics of nonchronic and chronic ulcers. These wounds show: (1) excessive production of cytokines, in particular three chemokines (KC/CXCL8, MCP‐1/CCL2, IP‐10/CXCL10), that may be key to the abnormal initiation and resolution of inflammation; (2) defective basement membranes, explaining blood vessel leakage and disruption of dermal/epidermal interactions; and (3) granulation tissue that contains high levels of Coll III, whereas Coll I is virtually absent and does not form fibrils. We also see major differences between nonchronic and chronic wounds, with the latter populated by bacterial films and producing eotaxin, a chemokine that attracts leukocytes that combat multicellular organisms (which biofilms can be considered to be). This new mouse model captures many defects observed in impaired and chronic human wounds and provides a vehicle to address their underlying cell and molecular mechanisms.

YOUNG INVESTIGATOR AWARD ARTICLE: Vascular endothelial growth factor promotes macrophage apoptosis through stimulation of tumor necrosis factor superfamily member 14 (TNFSF14/LIGHT)

Resolution of inflammation is critical for normal wound healing. Inflammation is prolonged and fails to resolve properly in chronic wounds. We used in vivo and in vitro approaches to show that vascular endothelial growth factor (VEGF) induces macrophage apoptosis and to delineate mechanisms involved in this process. VEGF inhibition during wound healing leads to an increased number of macrophages remaining in wounds, suggesting the involvement of VEGF in removal of these cells from the wound. If this effect has physiological relevance, it likely occurs via apoptosis. We show that VEGF increases apoptosis of macrophages in vitro using Annexin V‐FITC staining and caspase activation. Microarray analysis, reverse transcription‐polymerase chain reaction, and immunoblotting showed that VEGF increases the expression of tumor necrosis factor superfamily member 14 (TNFSF14/LIGHT) in macrophages. We also show that in macrophages LIGHT promotes apoptosis through the lymphotoxin β receptor. Moreover, inhibition of LIGHT prevents VEGF‐induced death, suggesting that LIGHT mediates VEGF‐induced macrophage apoptosis. Taken together, our results identify a novel role for VEGF and for LIGHT in macrophage apoptosis during wound healing, an event critical in the resolution of inflammation. This finding may lead to the development of new strategies to improve resolution of inflammation in problematic wounds.

Activation of sterol regulatory element‐binding proteins (SREBPs) is critical in IL‐8‐induced angiogenesis

Angiogenesis is essential in many physiological and pathological processes and can be stimulated by many different fators. To better understand and to manipulate this process more effectively, it would be beneficial to identify molecules common to the signaling pathways stimulated by different classes of angiogenic factors. Sterol regulatory element‐binding proteins (SREBPs) are involved in the metabolism of cholesterol and fatty acids, molecules that are critical in membrane biology, and hence, many of the processes involved in angiogenesis. Here, we show that angiogenic factors of different families, such as basic fibroblast growth factor, thrombin, and interleukin (IL)‐8, stimulate SREBP activation, whereas nonangiogenic factors, such as transforming growth factor‐β1, do not. We focused our detailed studies on IL‐8 in vitro and in vivo, as this chemokine is also involved in inflammation and hence, has the potential to be critical in inflammation‐induced angiogenesis, a process common to many diseases. Using human microvascular endothelial cells, a rabbit skin wound‐healing model, and the chorioallantoic membrane assay, we show that IL‐8 stimulates the activation of SREBP‐1 and ‐2, and this activation is specific and receptor‐mediated. SREBP activation leads to activation of RhoA through 3‐hydroxy‐3‐methylglutaryl CoA reductase. RhoA is a small guanosinetriphosphatase, important in cytoskeletal functions, which in turn, are critical in many of the cellular processes needed for angiogenesis. Given that diverse, angiogenic factors use different cell‐surface receptors, identification of this common step in the signal‐transduction pathway provides the opportunity for novel approaches for prevention and treatment of diseases involving abnormal angiogenesis.

Vascular endothelial growth factor promotes macrophage apoptosis through stimulation of tumor necrosis factor superfamily member 14 (TNFSF14/LIGHT)

Resolution of inflammation is critical for normal wound healing. Inflammation is prolonged and fails to resolve properly in chronic wounds. We used in vivo and in vitro approaches to show that vascular endothelial growth factor (VEGF) induces macrophage apoptosis and to delineate mechanisms involved in this process. VEGF inhibition during wound healing leads to an increased number of macrophages remaining in wounds, suggesting the involvement of VEGF in removal of these cells from the wound. If this effect has physiological relevance, it likely occurs via apoptosis. We show that VEGF increases apoptosis of macrophages in vitro using Annexin V‐FITC staining and caspase activation. Microarray analysis, reverse transcription‐polymerase chain reaction, and immunoblotting showed that VEGF increases the expression of tumor necrosis factor superfamily member 14 (TNFSF14/LIGHT) in macrophages. We also show that in macrophages LIGHT promotes apoptosis through the lymphotoxin β receptor. Moreover, inhibition of LIGHT prevents VEGF‐induced death, suggesting that LIGHT mediates VEGF‐induced macrophage apoptosis. Taken together, our results identify a novel role for VEGF and for LIGHT in macrophage apoptosis during wound healing, an event critical in the resolution of inflammation. This finding may lead to the development of new strategies to improve resolution of inflammation in problematic wounds.

YOUNG INVESTIGATOR AWARD ARTICLE: Vascular endothelial growth factor promotes macrophage apoptosis through stimulation of tumor necrosis factor superfamily member 14 (TNFSF14/LIGHT): VEGF-induced macrophage apoptosis is mediated by LIGHT

Resolution of inflammation is critical for normal wound healing. Inflammation is prolonged and fails to resolve properly in chronic wounds. We used in vivo and in vitro approaches to show that vascular endothelial growth factor (VEGF) induces macrophage apoptosis and to delineate mechanisms involved in this process. VEGF inhibition during wound healing leads to an increased number of macrophages remaining in wounds, suggesting the involvement of VEGF in removal of these cells from the wound. If this effect has physiological relevance, it likely occurs via apoptosis. We show that VEGF increases apoptosis of macrophages in vitro using Annexin V‐FITC staining and caspase activation. Microarray analysis, reverse transcription‐polymerase chain reaction, and immunoblotting showed that VEGF increases the expression of tumor necrosis factor superfamily member 14 (TNFSF14/LIGHT) in macrophages. We also show that in macrophages LIGHT promotes apoptosis through the lymphotoxin β receptor. Moreover, inhibition of LIGHT prevents VEGF‐induced death, suggesting that LIGHT mediates VEGF‐induced macrophage apoptosis. Taken together, our results identify a novel role for VEGF and for LIGHT in macrophage apoptosis during wound healing, an event critical in the resolution of inflammation. This finding may lead to the development of new strategies to improve resolution of inflammation in problematic wounds.