John M. Rhodes

The extracellular matrix and blood vessel formation: not just a scaffold.

•  Introduction •  ECM: composition and structure ‐  Collagen ‐  Laminin ‐  Fibronectin ‐  Elastin ‐  Nidogen ‐  Glycosaminoglycans ‐  Perlecan ‐  Syndecans ‐  Receptors for ECM molecules •  Evaluation of ECM molecules and their modulators in vessel formation ‐  Determination of essential ECM components of vessel formation ‐  Collagen‐I and laminins are important for vessel structural integrity and provide contrasting signals in angiogenesis ‐  The function of collagens in the blood vessel basement membrane ‐  Laminin α4 is a key molecule in basement membrane assembly, microvessel stability and maturation ‐  Fibronectin is essential for vascular development ‐  Perlecan and syndecans modulate growth factors in angiogenesis ‐  Fragments of ECM proteins regulate angiogenic processes ‐  Proteins involved in signalling through ECM molecules ‐  Thrombospondins ‐  CCN proteins •  ECM signalling in vessel formation: what next?

Suppression of RhoG activity is mediated by a syndecan 4–synectin–RhoGDI1 complex and is reversed by PKCα in a Rac1 activation pathway

Fibroblast growth factor 2 (FGF2) is a major regulator of developmental, pathological, and therapeutic angiogenesis. Its activity is partially mediated by binding to syndecan 4 (S4), a proteoglycan receptor. Angiogenesis requires polarized activation of the small guanosine triphosphatase Rac1, which involves localized dissociation from RhoGDI1 and association with the plasma membrane. Previous work has shown that genetic deletion of S4 or its adapter, synectin, leads to depolarized Rac activation, decreased endothelial migration, and other physiological defects. In this study, we show that Rac1 activation downstream of S4 is mediated by the RhoG activation pathway. RhoG is maintained in an inactive state by RhoGDI1, which is found in a ternary complex with synectin and S4. Binding of S4 to synectin increases the latters binding to RhoGDI1, which in turn enhances RhoGDI1s affinity for RhoG. S4 clustering activates PKCα, which phosphorylates RhoGDI1 at Ser96. This phosphorylation triggers release of RhoG, leading to polarized activation of Rac1. Thus, FGF2-induced Rac1 activation depends on the suppression of RhoG by a previously uncharacterized ternary S4–synectin–RhoGDI1 protein complex and activation via PKCα.

Regulation of Notch signaling by Drosophila heparan sulfate 3-O sulfotransferase

Heparan sulfate (HS) regulates the activity of various ligands and is involved in molecular recognition events on the cell surface and in the extracellular matrix. Specific binding of HS to different ligand proteins depends on the sulfation pattern of HS. For example, the interaction between antithrombin and a particular 3-O sulfated HS motif is thought to modulate blood coagulation. However, a recent study of mice defective for this modification suggested that 3-O sulfation plays other biological roles. Here, we show that Drosophila melanogaster HS 3-O sulfotransferase-b (Hs3st-B), which catalyzes HS 3-O sulfation, is a novel component of the Notch pathway. Reduction of Hs3st-B function by transgenic RNA interference compromised Notch signaling, producing neurogenic phenotypes. We also show that levels of Notch protein on the cell surface were markedly decreased by loss of Hs3st-B. These findings suggest that Hs3st-B is involved in Notch signaling by affecting stability or intracellular trafficking of Notch protein.

Syndecan 4 is required for endothelial alignment in flow and atheroprotective signaling

Significance Atherosclerosis, the major cause of death and illness in industrialized nations, develops in regions of arteries in which fluid flow patterns are disturbed and endothelial cells fail to align in the direction of flow. In contrast, regions of laminar flow in which cells are aligned are protected. The current work shows that the transmembrane proteoglycan syndecan 4 is required for endothelial cell alignment in the direction of flow and for the protective effect of high laminar flow, yet other flow responses are intact. The data therefore identify a role for syndecan 4 in flow direction sensing, show that sensing flow direction is separable from sensing flow magnitude, and provide new support for the key role of cell alignment in atheroprotection. Atherosclerotic plaque localization correlates with regions of disturbed flow in which endothelial cells (ECs) align poorly, whereas sustained laminar flow correlates with cell alignment in the direction of flow and resistance to atherosclerosis. We now report that in hypercholesterolemic mice, deletion of syndecan 4 (S4−/−) drastically increased atherosclerotic plaque burden with the appearance of plaque in normally resistant locations. Strikingly, ECs from the thoracic aortas of S4−/− mice were poorly aligned in the direction of the flow. Depletion of S4 in human umbilical vein endothelial cells (HUVECs) using shRNA also inhibited flow-induced alignment in vitro, which was rescued by re-expression of S4. This effect was highly specific, as flow activation of VEGF receptor 2 and NF-κB was normal. S4-depleted ECs aligned in cyclic stretch and even elongated under flow, although nondirectionally. EC alignment was previously found to have a causal role in modulating activation of inflammatory versus antiinflammatory pathways by flow. Consistent with these results, S4-depleted HUVECs in long-term laminar flow showed increased activation of proinflammatory NF-κB and decreased induction of antiinflammatory kruppel-like factor (KLF) 2 and KLF4. Thus, S4 plays a critical role in sensing flow direction to promote cell alignment and inhibit atherosclerosis.

Synectin/syndecan-4 regulate coronary arteriolar growth during development

Syndecan‐4 and its cytoplasmic binding partner, synectin, are known to play a role in FGF‐2 signaling and vascular growth. To determine their roles in coronary artery/arteriolar formation and growth, we compared syndecan‐4 and synectin null mice with their wild‐type counterparts. Image analysis of arterioles visualized by smooth muscle α‐actin immunostaining revealed that synectin −/− mice had lower arteriolar length and volume densities than wild‐type mice. As shown by electron microscopic analysis, arterioles from the two did not differ in morphology, including their endothelial cell junctions, and the organization and distribution of smooth muscle. Using micro‐computer tomography, we found that the size and branching patterns of coronary arteries (diameters > 50 μm) were similar for the two groups, a finding that indicates that the growth of arteries is not influenced by a loss of synectin. Syndecan‐4 null male mice also had lower arteriolar length densities than their gender wild‐type controls. However, female syndecan‐4 null mice were characterized by higher arteriolar length and volume densities than their gender‐matched wild‐type controls. Thus, we conclude that both synectin and syndecan‐4 play a role in arteriolar development, a finding that is consistent with previous evidence that FGF‐2 plays a role in coronary arterial growth. Moreover, our data reveal that gender influences the arteriolar growth response to syndecan‐4 but not to synectin. Developmental Dynamics 236:2004–2010, 2007.

Science and technology consortia in U.S. biomedical research: A paradigm shift in response to unsustainable academic growth

Modern-day bioscientific research is now at an economic crossroads. Specifically, the past 30 years have brought extraordinary advancements in biomedical knowledge, initiating the era of “personalized medicine”, therapies tailored specifically to individual patients’ genomic, epigenomic, and transcriptomic profiles, discovery of drugs based on computational analyses of massive datasets, and systems pharmacology (optimizing dosing and detection of adverse drug events). These have resulted in an exponential increase in our understanding of biological processes at the molecular, cellular, and organismal levels. Overall U.S. life expectancy (at birth) has risen from 74.7 years in 1985 to 78.7 years in 2010. Concurrently, the U.S. infant mortality rate declined by 42%, while mortality from heart disease and cancer decreased by 53% and 20%, respectively. Despite these monumental advances, the traditional manner in which

Challenging the Surgical Rodent Hindlimb Ischemia Model with the Miniinterventional Technique

PURPOSE To develop an interventional hindlimb ischemic model and compare its angiogenic effect versus surgical ligation (SL) and excision of the femoral artery in rats treated with transplantation of bone marrow mononuclear cells (MNCs) as an angiogenic stimulator. MATERIALS AND METHODS Forty-eight Lewis rats randomly received interventional embolization (IE) with hydrogel wire or SL and excision of the right femoral artery. Rodents were intraarterially transplanted with 1.5 × 10(7) MNCs in 500 μL medium from 24 isogenic donor rats. Functional and structural recovery was evaluated by laser Doppler imaging (LDI), cytokine/chemokine assay, and histologic staining. RESULTS In vivo microscopic images showed significantly dilated vasa vasorum around the embolized segment of the right femoral artery at 3 days compared with disorganized tissue structure in the SL group. However, the LDI index was significantly higher in the SL group at 3 days compared with the IE group. LDI did not significantly differ between the two groups at 2 weeks after transplantation. Cytokine assay showed higher levels of interleukin (IL)-1α and IL-18 in the SL group; the IE group had higher levels of interferon-γ, IL-6, IL-13, and granulocyte colony-stimulating factor. Histologic examination demonstrated inflammatory infiltration near the incision within nerve fibers with dilated capillaries, showing nerve degeneration in the SL group. At 2 weeks, histologic analysis demonstrated massive scarring under the skin spreading into the musculature in the SL group. CONCLUSIONS A minimally invasive hindlimb ischemia model has been successfully developed that preserves tissue integrity and minimizes inflammation and confounding factors in the early stages of angiogenesis and arteriogenesis.

Science and technology consortia in U.S. biomedical research

Modern-day bioscientific research is now at an economic crossroads. Specifically, the past 30 years have brought extraordinary advancements in biomedical knowledge, initiating the era of “personalized medicine”, therapies tailored specifically to individual patients’ genomic, epigenomic, and transcriptomic profiles, discovery of drugs based on computational analyses of massive datasets, and systems pharmacology (optimizing dosing and detection of adverse drug events). These have resulted in an exponential increase in our understanding of biological processes at the molecular, cellular, and organismal levels. Overall U.S. life expectancy (at birth) has risen from 74.7 years in 1985 to 78.7 years in 2010. Concurrently, the U.S. infant mortality rate declined by 42%, while mortality from heart disease and cancer decreased by 53% and 20%, respectively. Despite these monumental advances, the traditional manner in which

Science and technology consortia in U.S. biomedical research: A paradigm shift in response to unsustainable academic growth: Thoughts & Opinion

Modern-day bioscientific research is now at an economic crossroads. Specifically, the past 30 years have brought extraordinary advancements in biomedical knowledge, initiating the era of “personalized medicine”, therapies tailored specifically to individual patients’ genomic, epigenomic, and transcriptomic profiles, discovery of drugs based on computational analyses of massive datasets, and systems pharmacology (optimizing dosing and detection of adverse drug events). These have resulted in an exponential increase in our understanding of biological processes at the molecular, cellular, and organismal levels. Overall U.S. life expectancy (at birth) has risen from 74.7 years in 1985 to 78.7 years in 2010. Concurrently, the U.S. infant mortality rate declined by 42%, while mortality from heart disease and cancer decreased by 53% and 20%, respectively. Despite these monumental advances, the traditional manner in which