Michael G. Davis

DISSOCIATION OF VASOCONSTRICTOR-STIMULATED BASIC FIBROBLAST GROWTH FACTOR EXPRESSION FROM HYPERTROPHIC GROWTH IN CULTURED VASCULAR SMOOTH MUSCLE CELLS : RELEVANT ROLES OF PROTEIN KINASE C

Thromboxane A2 (TXA2) and angiotensin II (Ang II) stimulate vascular smooth muscle hypertrophy by upregulating endogenous synthesis of basic fibroblast growth factor (bFGF). Because mitogenic phorbol esters can also stimulate bFGF formation, we investigated the role of protein kinase C (PKC) in vascular smooth muscle cell (VSMC) bFGF formation and hypertrophy. Preliminary characterization of PKC isoform expression in VSMC by use of polymerase chain reaction identified PKC alpha, delta, epsilon, and zeta. Western analysis confirmed the presence of these isoforms in cultured VSMC lines and demonstrated downregulation of PKC alpha, delta, and epsilon by phorbol 12-myristate 13-acetate (PMA) but not TXA2 or Ang II. PKC activation with 100 nmol/L PMA stimulated VSMC mitogenesis measured as incorporation of [3H]leucine and [3H]thymidine and increased cell number. Like TXA2 and Ang II, PMA increased endogenous VSMC bFGF in a time-dependent manner, whereas an inactive phorbol ester had no such effect. Addition of an antisense oligodeoxynucleotide against bFGF prevented PMA-stimulated bFGF expression and inhibited PMA-stimulated growth, suggesting that bFGF synthesis is necessary for VSMC growth stimulated by PMA. To clarify the role of PKC in vasoconstrictor-stimulated VSMC production of bFGF and hypertrophy, PKC was down-regulated by prolonged exposure to PMA or was inhibited with calphostin C or staurosporine before the addition of TXA2 or Ang II. PKC inhibition prevented TXA2-stimulated and Ang II--stimulated VSMC hypertrophy without attenuating the observed increase in bFGF expression. Furthermore, PKC inhibition with calphostin C inhibited VSMC mitogenesis stimulated by exogenous bFGF.(ABSTRACT TRUNCATED AT 250 WORDS)

Tyrosine kinase inhibition prevents deformation-stimulated vascular smooth muscle growth.

The goal of this study was to determine the role of tyrosine phosphorylation in transducing deformation-stimulated vascular smooth muscle growth. Rat aorta-derived vascular smooth muscle cells were cultured on flexible silicone elastomer membranes and subjected to cyclic deformation (15 cycles per minute, deformed 2 seconds, relaxed 2 seconds). Deformation significantly increased proto-oncogene expression, [3H]thymidine incorporation, [3H]leucine incorporation, and cell number. Time course studies showed an 8-hour lag between initiation of cell deformation and onset of [3H]thymidine incorporation, with peak levels achieved after 18 to 24 hours. Western analysis of protein blots from deformed cells (10 minutes) demonstrated increased levels of phosphotyrosine-containing proteins having molecular weights of 110 to 130 and 70 to 80 kD. Deformation-stimulated tyrosine phosphorylation was prevented by the tyrosine kinase inhibitor Herbimycin A. Tyrosine kinase inhibition also prevented deformation-stimulated vascular smooth muscle cell growth as measured by [3H]thymidine incorporation. Cyclic deformation stimulates vascular smooth muscle proliferation through activation of tyrosine kinases. Inhibition of tyrosine phosphorylation is an effective means of preventing deformation-induced vascular smooth muscle growth in vitro.