Identification of cyclins A1, E1 and vimentin as downstream targets of heme oxygenase-1 in vascular endothelial growth factor-mediated angiogenesis

Angiogenesis is an essential physiological process and an important factor in disease pathogenesis. However, its exploitation as a clinical target has achieved limited success and novel molecular targets are required. Although heme oxygenase-1 (HO-1) acts downstream of vascular endothelial growth factor (VEGF) to modulate angiogenesis, knowledge of the mechanisms involved remains limited. We set out identify novel HO-1 targets involved in angiogenesis. HO-1 depletion attenuated VEGF-induced human endothelial cell (EC) proliferation and tube formation. The latter response suggested a role for HO-1 in EC migration, and indeed HO-1 siRNA negatively affected directional migration of EC towards VEGF; a phenotype reversed by HO-1 over-expression. EC from Hmox1(-/-) mice behaved similarly. Microarray analysis of HO-1-depleted and control EC exposed to VEGF identified cyclins A1 and E1 as HO-1 targets. Migrating HO-1-deficient EC showed increased p27, reduced cyclin A1 and attenuated cyclin-dependent kinase 2 activity. In vivo, cyclin A1 siRNA inhibited VEGF-driven angiogenesis, a response reversed by Ad-HO-1. Proteomics identified structural protein vimentin as an additional VEGF-HO-1 target. HO-1 depletion inhibited VEGF-induced calpain activity and vimentin cleavage, while vimentin silencing attenuated HO-1-driven proliferation. Thus, vimentin and cyclins A1 and E1 represent VEGF-activated HO-1-dependent targets important for VEGF-driven angiogenesis.National Heart and Lung Institute Foundation UK charity studentship: (Charity no. 1048073); National Institute for Health Research (NIHR); Biomedical Research Centre; Imperial College Healthcare NHS; Trust and Imperial College London

PKCε-CREB-Nrf2 signalling induces HO-1 in the vascular endothelium and enhances resistance to inflammation and apoptosis

Aims Vascular injury leading to endothelial dysfunction is a characteristic feature of chronic renal disease, diabetes mellitus, and systemic inflammatory conditions, and predisposes to apoptosis and atherogenesis. Thus, endothelial dysfunction represents a potential therapeutic target for atherosclerosis prevention. The observation that activity of either protein kinase C epsilon (PKCε) or haem oxygenase-1 (HO-1) enhances endothelial cell (EC) resistance to inflammation and apoptosis led us to test the hypothesis that HO-1 is a downstream target of PKCε. Methods and results Expression of constitutively active PKCε in human EC significantly increased HO-1 mRNA and protein, whereas conversely aortas or cardiac EC from PKCε-deficient mice exhibited reduced HO-1 when compared with wild-type littermates. Angiotensin II activated PKCε and induced HO-1 via a PKCε-dependent pathway. PKCε activation significantly attenuated TNFα-induced intercellular adhesion molecule-1, and increased resistance to serum starvation-induced apoptosis. These responses were reversed by the HO antagonist zinc protoporphyrin IX. Phosphokinase antibody array analysis identified CREB1(Ser133) phosphorylation as a PKCε signalling intermediary, and cAMP response element-binding protein 1 (CREB1) siRNA abrogated PKCε-induced HO-1 up-regulation. Likewise, nuclear factor (erythroid-derived 2)-like 2 (Nrf2) was identified as a PKCε target using nuclear translocation and DNA-binding assays, and Nrf2 siRNA prevented PKCε-mediated HO-1 induction. Moreover, depletion of CREB1 inhibited PKCε-induced Nrf2 DNA binding, suggestive of transcriptional co-operation between CREB1 and Nrf2. Conclusions PKCε activity in the vascular endothelium regulates HO-1 via a pathway requiring CREB1 and Nrf2. Given the potent protective actions of HO-1, we propose that this mechanism is an important contributor to the emerging role of PKCε in the maintenance of endothelial homeostasis and resistance to injury

Vascular protection during systemic inflammation; Investigation of the relationship between PKC epsilon and heme oxygenase-1

Background: Patients with chronic inflammatory diseases including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) demonstrate endothelial dysfunction. The inflammatory response triggered by endothelial injury leads to reduced NO biosynthesis, while increasing superoxide generation, monocyte adhesion and endothelial cell (EC) apoptosis. Apoptosis occurs preferentially at atherosclerosis prone sites, where endothelial erosion increases the risk of thrombosis and plaque development. Thus, chronic endothelial injury is a significant factor in the accelerated atherosclerosis associated with SLE and RA. Identifying the mechanisms underlying vascular injury and understanding innate cytoprotective pathways are essential for novel therapy development. Results: Known to play a role in cardiomyocyte ischemic preconditioning, protein kinase C (PKC) ε co-precipitates with stress-activated proteins and induces anti-apoptotic genes. We have now identified PKCε as an important regulator of cytoprotective responses in the vascular endothelium. We have shown that activation of PKCε induces expression of the cytoprotective enzyme HO-1 in human umbilical vein EC, using qRT-PCR and immunoblotting. This response is dependent upon gene transcription and de novo protein synthesis. A combined siRNA and immunohistochemical approach revealed a role for CREB and Nrf2 in PKCε-mediated HO-1 induction via a novel mechanism, which may involve positive feedback mechanisms and the dimerization of these two transcription factors. PKCε- dependent HO-1 expression is inducible by angiotensin II and able to protect against apoptosis and TNF-α- induced ICAM-1 upregulation. Furthermore, analysis of cardiac EC isolated from PKCε-/- and wild-type mice demonstrated altered expression of HO-1 in PKCε-/- EC and increased generation of reactive oxygen species. Conclusion: We have demonstrated a regulatory role for PKCε in the expression of the cytoprotective enzyme HO-1 in the vascular endothelium, and unearthed a novel signalling pathway involving CREB and Nrf2. The ultimate aim of this work is to identify novel therapeutic targets for treatment of endothelial dysfunction in systemic inflammatory diseases