Heme oxygenase (HO-1, encoded by Hmox1) is an inducible protein activated in systemic inflammatory conditions by oxidant stress. Vascular injury is characterized by a local reparative process with inflammatory components, indicating a potential protective role for HO-1 in arterial wound repair. Here we report that HO-1 directly reduces vasoconstriction and inhibits cell proliferation during vascular injury. Expression of HO-1 in arteries stimulated vascular relaxation, mediated by guanylate cyclase and cGMP, independent of nitric oxide. The unexpected effects of HO-1 on vascular smooth muscle cell growth were mediated by cell-cycle arrest involving p21Cip1. HO-1 reduced the proliferative response to vascular injury in vivo; expression of HO-1 in pig arteries inhibited lesion formation and Hmox1-/- mice produced hyperplastic arteries compared with controls. Induction of the HO-1 pathway moderates the severity of vascular injury by at least two adaptive mechanisms independent of nitric oxide, and is a potential therapeutic target for diseases of the vasculature.

The cyclin-dependent kinase inhibitor, p27(Kip1), which regulates cell cycle progression, is controlled by its subcellular localization and subsequent degradation. p27(Kip1) is phosphorylated on serine 10 (S10) and threonine 187 (T187). Although the role of T187 and its phosphorylation by Cdks is well-known, the kinase that phosphorylates S10 and its effect on cell proliferation has not been defined. Here, we identify the kinase responsible for S10 phosphorylation as human kinase interacting stathmin (hKIS) and show that it regulates cell cycle progression. hKIS is a nuclear protein that binds the C-terminal domain of p27(Kip1) and phosphorylates it on S10 in vitro and in vivo, promoting its nuclear export to the cytoplasm. hKIS is activated by mitogens during G(0)/G(1), and expression of hKIS overcomes growth arrest induced by p27(Kip1). Depletion of KIS using small interfering RNA (siRNA) inhibits S10 phosphorylation and enhances growth arrest. p27(-/-) cells treated with KIS siRNA grow and progress to S/G(2 )similar to control treated cells, implicating p27(Kip1) as the critical target for KIS. Through phosphorylation of p27(Kip1) on S10, hKIS regulates cell cycle progression in response to mitogens.

Abstract-As thrombin binding to the G protein-coupled proteinase activated receptor-1 (PAR-1) induces endothelial adhesivity to leukocytes through NF-B activation and intercellular adhesion molecule-1 (ICAM-1) expression, we determined the signaling pathways mediating the response. Studies showed that the heterotrimeric G proteins, G␣ q , and the G␤␥ dimer were key determinants of the PAR-1 agonist peptide (TFLLRNPNDK)-induced NF-B activation and ICAM-1 expression in endothelial cells. Cotransfection of RGS3T, a regulator of G-protein signaling that inhibits G␣ q , or ␣-transducin (G␣ t ), a scavenger of the G␤␥, markedly decreased NF-B activity induced by PAR-1 activation. We determined the downstream signaling targets activated by G␣ q and G␤␥ that mediate NF-B activation. Expression of the kinase-defective protein kinase C (PKC)-␦ mutant inhibited NF-B activation induced by the constitutively active G␣ q mutant, but had no effect on NF-B activity induced by G␤ 1 ␥ 2 . In related experiments, NF-B as well as ICAM-1 promoter activation induced by G␤ 1 ␥ 2 were inhibited by the expression of the dominant-negative mutant of 85-kDa regulatory subunit of PI 3-kinase; however, the expression of this mutant had no effect on the response induced by activated G␣

Abstract-Heme oxygenase (HO)-1 (encoded by Hmox1) catalyzes the oxidative degradation of heme to biliverdin and carbon monoxide. HO-1 is induced during inflammation and oxidative stress to protect tissues from oxidative damage.

Primary lung fibroblasts were isolated from patients with idiopathic pulmonary fibrosis (HIPF), from normal human lung tissue (NH), from rats treated with 75% oxygen and paraquat (PA), and from normal adult rats (NR). Serum-free media conditioned by each fibroblast strain were tested on the human A549 cell line (HIPF and NH media) or on primary alveolar epithelial cells (AEC) isolated from normal adult rats (PA or NR media). Over 20-h incubation, HIPF- or PA-conditioned media induced DNA fragmentation and significant decreases in total recoverable DNA and cell number of A549 or AEC, respectively; NH or NR media had no significant effect relative to serum-free unconditioned media. Apoptosis of A549 and AEC was detected by altered nuclear morphology and was confirmed by terminal deoxynucleotidyl transferase-mediated bio-dUTP nick end labeling. The endonuclease inhibitors 10 microM aurintricarboxylic acid and 50 microM zinc inhibited HIPF-induced apoptosis of A549 cells by 68 and 71%, respectively. Both apoptosis and necrosis were induced by HIPF and PA media in a concentration-dependent manner. These results suggest that altered fibroblasts emerging during fibrotic lung injury release a soluble factor(s) capable of inducing cell death and net loss of AEC.

Reactive oxygen species have been proposed to signal the activation of the transcription factor nuclear factor (NF)-kappaB in response to tumor necrosis factor (TNF)-alpha challenge. In the present study, we investigated the effects of H(2)O(2) and TNF-alpha in mediating activation of NF-kappaB and transcription of the intercellular adhesion molecule (ICAM)-1 gene. Northern blot analysis showed that TNF-alpha exposure of human dermal microvascular endothelial cells (HMEC-1) induced marked increases in ICAM-1 mRNA and cell surface protein expression. In contrast, H(2)O(2) added at subcytolytic concentrations failed to activate ICAM-1 expression. Challenge with H(2)O(2) also failed to induce NF-kappaB-driven reporter gene expression in the transduced HMEC-1 cells, whereas TNF-alpha increased the NF-kappaB-driven gene expression approximately 10-fold. Gel supershift assay revealed the presence of p65 (Rel A), p50, and c-Rel in both H(2)O(2)- and TNF-alpha-induced NF-kappaB complexes bound to the ICAM-1 promoter, with the binding of the p65 subunit being the most prominent. In vivo phosphorylation studies, however, showed that TNF-alpha exposure induced marked phosphorylation of NF-kappaB p65 in HMEC-1 cells, whereas H(2)O(2) had no effect. These results suggest that reactive oxygen species generation in endothelial cells mediates the binding of NF-kappaB to nuclear DNA, whereas TNF-alpha generates additional signals that induce phosphorylation of the bound NF-kappaB p65 and confer transcriptional competency to NF-kappaB.

The cyclin-dependent kinase inhibitors are key regulators of cell cycle progression. Although implicated in carcinogenesis, they inhibit the proliferation of a variety of normal cell types, and their role in diverse human diseases is not fully understood. Here, we report that p27 Kip1 plays a major role in cardiovascular disease through its effects on the proliferation of bone marrow-derived (BM-derived) immune cells that migrate into vascular lesions. Lesion formation after mechanical arterial injury was markedly increased in mice with homozygous deletion of p27 Kip1 , characterized by prominent vascular infiltration by immune and inflammatory cells. Vascular occlusion was substantially increased when BM-derived cells from p27 -/-mice repopulated vascular lesions induced by mechanical injury in p27 +/+ recipients, in contrast to p27 +/+ BM donors. To determine the contribution of immune cells to vascular injury, transplantation was performed with BM derived from RAG -/-and RAG +/+ mice. RAG +/+ BM markedly exacerbated vascular proliferative lesions compared with what was found in RAG -/-donors. Taken together, these findings suggest that vascular repair and regeneration is regulated by the proliferation of BM-derived hematopoietic and nonhematopoietic cells through a p27 Kip1 -dependent mechanism and that immune cells largely mediate these effects.

We investigated the mechanisms by which elevated intracellular cAMP concentration inhibits the thrombin-induced ICAM-1 expression in endothelial cells. Exposure of human umbilical vein endothelial cells to forskolin or dibutyryl cAMP, which increase intracellular cAMP by separate mechanisms, inhibited the thrombin-induced ICAM-1 expression. This effect of cAMP was secondary to inhibition of NF-kappaB activity, the key regulator of thrombin-induced ICAM-1 expression in endothelial cells. The action of cAMP occurred downstream of IkappaBalpha degradation and was independent of NF-kappaB binding to the ICAM-1 promoter. We observed that cAMP interfered with thrombin-induced phosphorylation of NF-kappaB p65 (RelA) subunit, a crucial event promoting the activation of the DNA-bound NF-kappaB. Because p38 MAPK can induce transcriptional activity of RelA/p65 without altering the DNA binding function of NF-kappaB, we addressed the possibility that cAMP antagonizes thrombin-induced NF-kappaB activity and ICAM-1 expression by preventing the activation of p38 MAPK. We observed that treating cells with forskolin blocked the activation of p38 MAPK, and inhibition of p38 MAPK interfered with phosphorylation of RelA/p65 induced by thrombin. Our data demonstrate that increased intracellular cAMP concentration in endothelial cells prevents thrombin-induced ICAM-1 expression by inhibiting p38 MAPK activation, which in turn prevents phosphorylation of RelA/p65 and transcriptional activity of the bound NF-kappaB.