Transient reexpression of an embryonic autonomous growth phenotype by adult carotid artery smooth muscle cells after vascular injury.

High rates of vascular smooth muscle cell (SMC) replication are observed, at least transiently, after injury to the arterial wall and contribute to the formation of a neointima. Neutralizing antibodies designed to inhibit growth of SMC have only been variably successful in inhibiting neointima formation, raising the possibility that neointimal cell proliferation involves unique growth mechanisms. This study examined the possibility that SMC isolated from injured rat carotid arteries would express an autonomous, mitogen-independent growth phenotype similar to that utilized by embryonic vascular SMC during periods of rapid growth. We found that primary cultures of SMC isolated 7 and 14 days after injury, times at which high in vivo replication rates were observed, demonstrated high intrinsic DNA synthetic rates compared to SMC isolated from uninjured arteries or at 2, 4, 21, and 28 days after injury where in vivo replication rates were far less. Subcultured SMC isolated from 7-day injured vessels (Neo7 SMC) exhibited a stable, autonomous growth phenotype, did not secrete detectable mitogenic activity, and had decreased alpha-actin and myosin expression compared to mitogen-dependent SMC. Heterokaryons constructed between autonomous Neo7 SMC and mitogen-dependent SMC exhibited a mitogen-dependent growth phenotype suggesting that nonautonomous SMC produce factors that actively inhibit autonomous growth. In contrast, heterokaryons constructed between Neo7 SMC and autonomous embryonic SMC retained an autonomous growth phenotype. We examined the expression of known tumor suppressors to determine if any of these factors played a role in inhibiting SMC autonomous growth. p27, p53, pRb, and PTEN were abundantly expressed by Neo7 SMC and e17 SMC under both basal and serum stimulated conditions. The data suggest that the mechanisms driving SMC replication during neointimal formation are self-driven and self-regulated, and that at specific times after injury, SMC escape normal growth suppressive mechanisms through the loss of intracellular growth suppressor activity.

In vitro studies of a bradykinin B1/B2 antagonist linked to a human neutrophil elastase inhibitor; a heterodimer for the treatment of inflammatory disorders.

Inflammatory disorders typically have a complex etiology and involve a multitude of inflammatory mediators, and hence, a polytherapeutic approach to these diseases would seem appropriate. In certain chronic inflammatory conditions, we believe that bradykinin (BK) and human neutrophil elastase (HNE) are cooperatively involved. We have previously synthesized compounds with inhibitory activity toward both the BK B2 receptor and HNE. The present study describes single compounds designed to incorporate HNE inhibitory activity and BK B1 and B2 antagonist activity. A proprietary HNE inhibitor (HNEI, CP-955) was directly linked via amide bond formation to a peptide-based combined BK B1/B2 antagonist (B-9430). Three compounds were made using different linking positions in the antagonist peptide. For all compounds, B1 and B2 receptor binding in human cloned receptors was at least 10-fold less than that of B-9430, whereas in the in vitro guinea pig ileum B2 receptor functional assay, the compounds had potencies equivalent to B-9430. Compound I was found to have a fourfold increase in HNEI activity compared with CP-955, whereas compounds II and III were inactive. These data clearly demonstrate that it is possible to retain BK B1/B2 receptor antagonist and HNE activity in a heterodimer.

Evidence of a bradykinin B1 receptor in human ileum: pharmacological comparison to the rabbit aorta B1 receptor.

Bradykinin B1 receptors have been identified in a limited number of human tissues and may have implications in pathological states of chronic inflammation. In the present study, longitudinal strips of postmortem human ileum displayed a strong contractile response to the B2 receptor agonist, bradykinin (EC50 = 7.0 nM). Noninduced ileum strips contracted only to high concentrations (1 and 10 microM) of the B1 receptor agonists, des-Arg9-BK and Lys0des-Arg9-BK. After incubation overnight at 37 degrees C the potency of des-Arg9-BK and Lys0des-Arg9-BK dramatically increased (EC50 = 183 and 13.2 nM, respectively). The increase in B1 agonist potency was inhibited by the protein synthesis inhibitor, puromycin. Similarly, rabbit aorta strips displayed a protein synthesis-dependent induction of the B1 agonist response. Incubated human ileum and rabbit aorta exhibited a reproducible response to des-Arg9-BK over time, whereas responses to Lys0des-Arg9-BK were not reproducible, having reduced potency and magnitude over time. Lys0[Leu8]des-Arg9-BK was a more potent antagonist at the B1 receptor in both tissues compared with [Leu8]des-Arg9-BK. The B2 antagonist, HOE-140, was a very weak inhibitor of the B1 response in human ileum and inactive in rabbit aorta. In conclusion, incubation of isolated human ileum overnight induces expression of a B1 receptor through a mechanism that depends on de novo protein synthesis. The potency profile of selected B1 agonists and antagonists indicates pharmacological similarities between the inducible B1 receptor in both the human ileum and rabbit aorta.