The small heat shock-related protein 20 (HSP20) is present in four isoforms in bovine carotid artery smooth muscles. Three of the isoforms are phosphorylated and one is not. Increases in the phosphorylation of two isoforms of HSP20 (isoform 3, pI 5.9; and 8, pI 5.7) are associated with cyclic nucleotide-dependent relaxation of bovine carotid artery smooth muscles. Increases in the phosphorylation of another isoform (isoform 4, pI 6.0) are associated with phorbol ester-induced contraction of bovine carotid artery smooth muscles. In this investigation we determined that isoforms 3 and 8 are phosphorylated on Ser 16 of the HSP20 molecule during activation of cAMP-dependent signaling pathways. Phosphorylation state-specific antibodies produced against a peptide containing phosphorylated Ser 16 recognized isoforms 3 and 8 but not isoform 4. In human vascular tissue, only isoform 3 is present. Incubation of transiently permeabilized strips of bovine carotid artery smooth muscle with synthetic peptides in which Ser 16 is phosphorylated, inhibits contractile responses to high extracellular KCl and to serotonin. These data suggest that phosphorylation of HSP20 on Ser 16 modulates cAMP-dependent vasorelaxation.A major phosphorylation event that occurs with cyclic nucleotide-dependent relaxation of vascular smooth muscle is an increase in the phosphorylation of two 20-kDa proteins (isoform 3, pI 5.9; and 8, pI 5.7) (1-3). We recently identified these 20-kDa phosphoproteins as different phosphorylated forms of a small heat shock-related protein, HSP20 1 (4). In addition, HSP20 can be phosphorylated in vitro by both cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) (4). HSP20 is also phosphorylated during endothelialdependent vasorelaxation of isolated segments of bovine carotid artery smooth muscle (5).In a vascular smooth muscle, umbilical artery smooth muscle, that is refractory to cyclic nucleotide-dependent vasorelaxation, there is no significant increase in the phosphorylation of HSP20 in response to activation of PKA or PKG (2). HSP20 is present in umbilical artery smooth muscle and can be phosphorylated by PKA in vitro using homogenates of umbilical smooth muscle (6). Taken together, these data support a role for phosphorylated HSP20 in mediating cyclic nucleotide-dependent vasorelaxation.Histamine and phorbol ester-induced contractions of bovine carotid artery smooth muscle are associated with an increase in the phosphorylation of another 20-kDa protein (isoform 4, pI 6.0) (1). The subsequent activation of cyclic nucleotide-dependent signaling pathways leads to a decrease in the phosphorylation of isoform 4. This 20-kDa protein is immunoreactive with specific polyclonal antibodies raised against HSP20 (4). Thus, increases in the phosphorylation of this isoform of HSP20 are associated with smooth muscle contraction and decreases are associated with activation of cyclic nucleotide-dependent signaling pathways.The purpose of this investigation was to determine the specific site on the HSP20 mo...

These results confirm our hypothesis that the source of cells resulting in venous stenosis formation is derived from the adventitia and media, with cell migration being greatest within the first two weeks after graft placement with translocation of these cells into the intima at four weeks. MMP-2 activity peaks at day seven in the adventitia and again at days 19 to 26 in the intima. A key to limiting venous stenosis formation may lie in inhibiting MMP-2 by adventitial and medial targeting.