Hemopexin promotes angiogenesis via up-regulating HO-1 in rats after cerebral ischemia-reperfusion injury.

BACKGROUND: Ischemia-reperfusion (I/R) is a critical pathophysiological change of ischemic stroke. Heme-oxygenase-1 (HO-1) is a rate-limiting enzyme of eliminating excessive free heme by combining with hemopexin (HPX), a plasma protein contributing to alleviating infarct size due to ischemia stroke. This study was to investigate whether HPX could improve angiogenesis after cerebral ischemia-reperfusion via up-regulating HO-1. METHODS: Rats were randomly divided into five groups: sham, MCAO, MCAO + Vehicle, MCAO + HPX and MCAO + HPX + protoporphyrin IX (ZnPPIX, an HO-1 inhibitor). Cerebral I/R was induced by MCAO. Saline, vehicle, HPX and HPX + ZnPPIX were respectively given to MCAO group, MCAO + Vehicle group, MCAO + HPX group and MCAO + HPX + ZnPPIX group at the moment after reperfusion by intracerebroventricular injection. Neurological behavioral scores(NBS) was assessed at 24 h and 7d after I/R. Real-time polymerase chain reaction (RT-PCR) was used to analyze the mRNA level of HO-1. Angiogenesis in penumbra area was assessed by immunofluorescence detection at 7d after I/R. Serum endothelial nitric oxide synthase (eNOS) was assessed by enzyme linked immunosorbent assay (ELISA) at 24 h and 7d after I/R. RESULTS: Compared with sham group, the NBS and the mRNA levels of HO-1 at 24 h and 7d after I/R in MCAO group decreased notably (P < 0.05), the new vessel density in ischemia penumbra increased notably at 7d after I/R (P < 0.05), the serum eNOS level increased at 24 h and 7d after I/R (P < 0.05). MCAO group and MCAO + Vehicle group showed no significant differences (P > 0.05). In the MCAO + HPX group, compared with MCAO + Vehicle group, the NBS and the mRNA levels of HO-1 increased drastically at 24 h and 7d after I/R (P < 0.05), the new vessel density in ischemia penumbra increased significantly at 7d after I/R (P < 0.05), the serum eNOS level at 24 h and 7d after I/R ascended notably (P < 0.05). Compared with MCAO + HPX group, the NBS assessment, new vessel density and serum eNOS level decreased at corresponding time points after I/R in MCAO + HPX+ ZnPPIX group (P < 0.05). CONCLUSION: HPX can promote angiogenesis after cerebral ischemia-reperfusion injury in rats via up-regulating HO-1.

Apolipoprotein E-/- Mice Lacking Hemopexin Develop Increased Atherosclerosis via Mechanisms That Include Oxidative Stress and Altered Macrophage Function.

OBJECTIVE: We previously reported that hemopexin (Hx), a heme scavenger, is significantly increased and associated with proinflammatory high-density lipoprotein under atherogenic conditions. Although it is established that Hx together with macrophages plays a role in mitigating oxidative damage, the role of Hx in the development of atherosclerosis is unknown. APPROACH AND RESULTS: We used Hx and apoE double-knockout mice (HxE(-/-)) to determine the role of Hx in the development of atherosclerosis. HxE(-/-) mice had significantly more free heme, reactive oxygen species, and proinflammatory high-density lipoprotein in their circulation, when compared with control apoE(-/-) mice. Atherosclerotic plaque area (apoE(-/-)=9.72±2.5×10(4) µm(2) and HxE(-/-)=27.23±3.6×10(4) µm(2)) and macrophage infiltration (apoE(-/-)=38.8±5.8×10(3) µm(2) and HxE(-/-)=103.4±17.8×10(3) µm(2)) in the aortic sinus were significantly higher in the HxE(-/-) mice. Atherosclerotic lesions in the aortas were significantly higher in the HxE(-/-) mice compared with apoE(-/-) mice. Analysis of polarization revealed that macrophages from HxE(-/-) mice were more M1-like. Ex vivo studies demonstrated that HxE(-/-) macrophage cholesterol efflux capacity was significantly reduced when compared with apoE(-/-) mice. Injection of human Hx into HxE(-/-) mice reduced circulating heme levels and human Hx pretreatment of naive bone marrow cells ex vivo resulted in a shift from M1- to M2-like macrophages. CONCLUSIONS: We conclude that Hx plays a novel protective role in alleviating heme-induced oxidative stress, improving inflammatory properties of high-density lipoprotein, macrophage phenotype and function, and inhibiting the development of atherosclerosis in apoE(-/-) mice.

Local intracerebral delivery of endogenous inhibitors by osmotic minipumps effectively suppresses glioma growth in vivo.

The systemic administration of endogenous inhibitors significantly reduced the growth of human glioma in vivo, but required the production of a large amount of biologically active protein. In this study we reduced the amount of protein needed and optimized the therapeutical response by delivering the endogenous inhibitors locally into the brain by osmotic minipumps. Human hemopexin fragment of MMP-2 or COOH-terminal fragment of platelet factor-4 were delivered locally and continuously into the brain of mice implanted intracranially with glioma cells, by osmotic minipumps connected to an intracranial catheter. Local delivery of human hemopexin fragment of MMP-2 and COOH-terminal fragment of platelet factor-4 significantly inhibited the growth of well-established malignant glioma in nude and BALB/C mice. When the inhibitors were given at the same concentration, the efficacy of the local delivery was much higher than that reached with the systemic administration, both when the inhibitor was administered daily or continuously by s.c. minipumps. Moreover, the local delivery reduced the amount of protein needed to reach a significant therapeutic response. Intracerebral delivery maintained a long-term control of glioma growth and inhibited glioma recurrence in a surgical resection model. Treatment showed no side effects. Histochemical analysis of tumors showed that the tumor growth inhibition was the result of a decrease in tumor vasculature and a change in tumor vessel morphology. Our data demonstrate that local intracerebral delivery of endogenous inhibitors effectively inhibits malignant glioma growth and reduces the amount of protein needed to reach a therapeutical response.