Background: Adiponectin has vascular protective actions and is bound by T-cadherin. Results: T-cadherin-deficient mice lack skeletal muscle tissue-resident adiponectin and display impaired revascularization that is not improved by treatment with exogenous adiponectin. Conclusion: Expression of T-cadherin is critical for revascularization actions of adiponectin in vitro and in vivo.Significance: The T-cadherin/adiponectin interaction is important for vascular homeostasis.

Vessel-like networks are quickly formed in subcutaneous FGF2-supplemented Matrigel plugs by two cell types: NG2+ pericytes and F4/80+ macrophages. Although not detected in these networks until 7 days after plug implantation, the appearance of CD31+ endothelial cells marks the onset of vessel perfusion and the establishment of mature vessel morphology, with endothelial cells invested tightly by pericytes and more loosely by macrophages. Evidence that mature vessels develop from pericyte/macrophage networks comes from experiments in which 5-day plugs are transplanted into EGFP+ recipients and allowed to mature. Fewer than 5% of pericytes in mature vessels are EGFP+ in this paradigm,demonstrating their presence in the networks prior to plug transplantation. Endothelial cells represent the major vascular cell type recruited during later stages of vessel maturation. Bone marrow transplantation using EGFP+ donors establishes that almost all macrophages and more than half of the pericytes in Matrigel vessels are derived from the bone marrow. By contrast, only 10% of endothelial cells exhibit a bone marrow origin. The vasculogenic, rather than angiogenic, nature of this neovascularization process is unique in that it is initiated by pericyte and macrophage progenitors, with endothelial cell recruitment occurring as a later step in the maturation process.

Pericytes play critical roles in the development, maturation and remodeling of blood vessels, and in the central nervous system (CNS), evidence suggests that pericytes also regulate blood flow and form an integral part of the blood-brain barrier. The study of this important cell type has been hampered by the lack of any pericyte-specific marker and by the difficulty of culturing pericytes in adequate numbers to high purity. Here we present a novel yet simple approach to isolate and culture large numbers of pericytes from the mouse CNS that nevertheless leads to very pure pericyte cultures. In our method, vascular cells obtained from adult mice brains are cultured initially under conditions optimized for endothelial cells, but after two passages switched to a medium optimized for pericyte growth. After growing the cells for 1-2 additional passages we obtained a largely homogeneous population of cells that expressed the pericyte markers NG2, PDGF -receptor, and CD146, but were negative for markers of endothelial cells (CD31), microglia (Mac-1) and astrocytes (GFAP). Under these conditions, pericytes could be grown to high passage number, and were maintained highly pure and largely undifferentiated, as determined by antigen expression profile and low levels of -SMA expression, a marker of pericyte differentiation. Furthermore, switching the cells from pericyte medium into DMEM containing 10% FBS promoted -SMA expression, demonstrating that high passage pericytes could still differentiate. Thus, we provide an alternative approach to the culture of CNS pericytes that is easy to establish and provides large numbers of highly pure pericytes for extended periods of time. This system should provide others working in the pericyte field with a useful additional tool to study the behavior of this fascinating cell type.

During cell spreading, binding of actin-organizing proteins to acidic phospholipids and phosphorylation are important for localization and activity of these proteins at nascent cell-matrix adhesion sites. Here, we report on a transient interaction between the lipid-dependent protein kinase C␣ and vinculin, an early component of these sites, during spreading of HeLa cells on collagen. In vitro binding of protein kinase C␣ to vinculin tail was found dependent on free calcium and acidic phospholipids but independent of a functional kinase domain. The interaction was enhanced by conditions that favor the oligomerization of vinculin. Phosphorylation by protein kinase C␣ reached 1.5 mol of phosphate/mol of vinculin tail and required the C-terminal hydrophobic hairpin, a putative phosphatidylinositol 4,5-bisphosphate-binding site. Mass spectroscopy of peptides derived from in vitro phosphorylated vinculin tail identified phosphorylation of serines 1033 and 1045. Inhibition of C-terminal phospholipid binding at the vinculin tail by mutagenesis or deletion reduced the rate of phosphorylation to <50%. We suggest a possible mechanism whereby phospholipid-regulated conformational changes in vinculin may lead to exposure of a docking site for protein kinase C␣ and subsequent phosphorylation of vinculin and/or vinculin interaction partners, thereby affecting the formation of cell adhesion complexes.