The cytochrome P450 arachidonic acid epoxygenase metabolites, the epoxyeicosatrienoic acids (EETs) are powerful, nonregioselective, stimulators of cell proliferation. In this study we compared the ability of the four EETs (5,6-, 8,9-, 11,12-, and 14,15-EETs) to regulate endothelial cell proliferation in vitro and angiogenesis in vivo and determined the molecular mechanism by which EETs control these events. Inhibition of the epoxygenase blocked serum-induced endothelial cell proliferation, and exogenously added EETs rescued cell proliferation from epoxygenase inhibition. Studies with selective ERK, p38 MAPK, or PI3K inhibitors revealed that whereas activation of p38 MAPK is required for the proliferative responses to 8,9-and 11,12-EET, activation of PI3K is necessary for the cell proliferation induced by 5,6-and 14,15-EET. Among the four EETs, only 5,6-and 8,9-EET are capable of promoting endothelial cell migration and the formation of capillary-like structures, events that are dependent on EET-mediated activation of ERK and PI3K. Using subcutaneous sponge models, we showed that 5,6-and 8,9-EET are pro-angiogenic in mice and that their neo-vascularization effects are enhanced by the co-administration of an inhibitor of EET enzymatic hydration, presumably because of reduced EET metabolism and inactivation. These studies identify 5,6-and 8,9-EET as powerful and selective angiogenic lipids, provide a functional link between the EET proliferative chemotactic properties and their angiogenic activity, and suggest a physiological role for them in angiogenesis and de novo vascularization.

Integrins control many cell functions, including generation of reactive oxygen species (ROS) and regulation of collagen synthesis. Mesangial cells, found in the glomerulus of the kidney, are able to produce large amounts of ROS via the NADPH oxidase. We previously demonstrated that integrin ␣1-null mice develop worse fibrosis than wild-type mice following glomerular injury and this is due, in part, to excessive ROS production by ␣1-null mesangial cells. In the present studies, we describe the mechanism whereby integrin ␣1-null mesangial cells produce excessive ROS. Integrin ␣1-null mesangial cells have constitutively increased basal levels of activated Rac1, which result in its increased translocation to the cell membrane, excessive ROS production, and consequent collagen IV deposition. Basal Rac1 activation is a direct consequence of ligand-independent increased epidermal growth factor receptor (EGFR) phosphorylation in ␣1-null mesangial cells. Thus, our study demonstrates that integrin ␣1␤1-EGFR cross talk is a key step in negatively regulating Rac1 activation, ROS production, and excessive collagen synthesis, which is a hallmark of diseases characterized by irreversible fibrosis.Integrin ␣1␤1, a major collagen binding receptor, is expressed in different cell types, including fibroblasts (45), endothelial cells (8), and mesangial cells in the glomerulus of the kidney (30, 53). Integrin ␣1␤1 confers the ability of cells to bind to collagenous substrata, including collagens I and IV (4, 45), and to proliferate on these substrata (45). Moreover, cells expressing integrin ␣1␤1 sense extracellular levels of collagen and downregulate its synthesis at both transcriptional and translational levels (4,14). Finally, we demonstrated that integrin ␣1␤1 also downregulates the production of reactive oxygen species (ROS) (4, 58).Following renal injury, mice lacking integrin ␣1␤1 develop more extensive glomerular fibrosis characterized by excessive accumulation of collagen type IV compared to wild-type (WT) mice (4, 58). Increased fibrosis is due to both a direct effect of the lack of integrin ␣1␤1-mediated downregulation of collagen IV synthesis and excessive ROS production by ␣1-null mesangial cells.Constitutive production of ROS by mesangial cells, a major cell type found in the glomerulus of the kidney, originates from an intrinsic NADPH oxidase (26, 48) that normally functions at a low level and increases in response to inflammatory stimuli, high glucose, or stress (25,34,35,37,55). The NADPH oxidase, highly characterized in phagocytes, is a multicomponent enzyme complex that consists of the membrane-bound cytochrome b 558 (p22 phox and gp91 phox ) and cytoplasmic proteins (p40 phox , p47 phox , p67 phox ) that translocate to the membrane following cellular stimulation to produce superoxide (3, 9, 47). A multicomponent phagocyte-like NADPH oxidase is also a major source of ROS in many nonphagocytic cells, including mesangial cells. In the phagocyte-like NADPH oxidase, the catalytic subunits are termed Nox proteins, with ...

Integrin ␣1␤1, the major collagen type IV receptor, is expressed by endothelial cells and plays a role in both physiologic and pathologic angiogenesis. Because the molecular mechanisms whereby this collagen IV receptor mediates endothelial cell functions are poorly understood, truncation and point mutants of the integrin ␣1 subunit cytoplasmic tail (amino acids 1137-1151) were generated and expressed into ␣1-null endothelial cells. We show that ␣1-null endothelial cells expressing the ␣1 subunit, which lacks the entire cytoplasmic tail (mutant ␣1-1136) or expresses all the amino acids up to the highly conserved GFFKR motif (mutant ␣1-1143), have a similar phenotype to parental ␣1-null cells. Pro 1144 and Leu 1145 were shown to be necessary for ␣1␤1-mediated endothelial cell proliferation; Lys 1146 for adhesion, migration, and tubulogenesis and Lys 1147 for tubulogenesis.Integrin ␣1␤1-dependent endothelial cell proliferation is primarily mediated by ERK activation, whereas migration and tubulogenesis require both p38 MAPK and PI3K/ Akt activation. Thus, distinct amino acids distal to the GFFKR motif of the ␣1 integrin cytoplasmic tail mediate activation of selective downstream signaling pathways and specific endothelial cell functions. (Blood. 2008;112:3242-3254)

The collagen binding integrins α1β1 and α2β1 have profoundly different functions, yet they are often co-expressed in epithelial cells. When both integrins are expressed in the same cell, it has been suggested that α1β1 negatively regulates integrin α2β1 dependent functions. In this study we utilized murine ureteric bud (UB) epithelial cells, which express no functionally detectable levels of endogenous integrins α1β1 and α2β1, to determine the mechanism whereby this regulation occurs. We demonstrate that UB cells expressing integrin α2β1, but not α1β1 adhere, migrate and proliferate on collagen I as well as form cellular cords in 3D collagen I gels. Substitution of the transmembrane domain of the integrin α2 subunit with that of α1 results in decreased cell adhesion, migration and cord formation. In contrast, substitution of the integrin α2 cytoplasmic tail with that of α1, decreases cell migration and cord formation, but increases proliferation. When integrin α1 and α2 subunits are co-expressed in UB cells, the α1 subunit negatively regulates integrin α2β1-dependent cord formation, adhesion and migration and this inhibition requires expression of both α1 and α2 tails. Thus, we provide evidence that the transmembrane and cytoplasmic domains of the α2 integrin subunit, as well as the α1 integrin subunit, regulate integrin α2β1 cell function.

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