622. Regulation of AAV Mediated VEGF Gene Expression by Hypoxia Response Element and Tissue Specific Promoter in the Ischemic Mouse Heart

AbstractNeural stem cell (NSCs) transplantation has been one of the hot topics in the repair of spinal cord injury (SCI). Fibroblast growth factor (FGF) is considered a promising nerve injury therapy after SCI. However, owing to a hostile hypoxia condition in SCI, there remains a challenging issue in implementing these tactics to repair SCI. In this report, we used adeno-associated virus 2 (AAV2), a prototype AAV used in clinical trials for human neuron disorders, basic FGF (bFGF) gene under the regulation of hypoxia response element (HRE) was constructed and transduced into NSCs to yield AAV2-5HRE-bFGF-NSCs. Our results showed that its treatment yielded temporally increased expression of bFGF in SCI, and improved scores of functional recovery after SCI compared to vehicle control (AAV2-5HRE-NSCs) based on the analyses of the inclined plane test, Basso–Beattie–Bresnahan (BBB) scale and footprint analysis. Mechanistic studies showed that AAV2-5HRE-bFGF-NSCs treatment increased the expression of neuron-specific neuronal nuclei protein (NeuN), neuromodulin GAP43, and neurofilament protein NF200 while decreased the expression of glial fibrillary acidic protein (GFAP) as compared to the control group. Further, the expressions of autophagy-associated proteins LC3-II and Beclin 1 were decreased, whereas the expression of P62 protein was increased in AAV2-5HRE-bFGF-NSCs treatment group. Taken together, our data indicate that AAV2-5HRE-bFGF-NSCs treatment improved the recovery of SCI rats, which is accompanied by evidence of nerve regeneration, and inhibition of SCI-induced glial scar formation and cell autophagy. Thus, this study represents a step forward towards the potential use of AAV2-5HRE-bFGF-NSCs for future clinical trials of SCI repair.

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Hypoxia response element of the human vascular endothelial growth factor gene mediates transcriptional regulation by nitric oxide: control of hypoxia-inducible factor-1 activity by nitric oxide

Blood ◽

10.1182/blood.v95.1.189 ◽

2000 ◽

Vol 95 (1) ◽

pp. 189-197 ◽

Cited By ~ 292

Author(s):

Hideo Kimura ◽

Alessandro Weisz ◽

Yukiko Kurashima ◽

Kouichi Hashimoto ◽

Tsutomu Ogura ◽

...

Keyword(s):

Nitric Oxide ◽

Vascular Endothelial Growth Factor ◽

Growth Factor ◽

Hypoxia Inducible Factor ◽

Vascular Endothelial ◽

Hypoxia Response Element ◽

Response Element ◽

Hypoxia Response ◽

Hypoxia Inducible Factor 1 ◽

Endothelial Growth Factor

Abstract Nitric oxide (NO) regulates production of vascular endothelial growth factor (VEGF) by normal and transformed cells. We demonstrate that NO donors may up-regulate the activity of the human VEGF promoter in normoxic human glioblastoma and hepatoma cells independent of a cyclic guanosine monophosphate–mediated pathway. Deletion and mutation analysis of the VEGF promoter indicates that the NO-responsive cis-elements are the hypoxia-inducible factor-1 (HIF-1) binding site and an adjacent ancillary sequence that is located immediately downstream within the hypoxia-response element (HRE). This work demonstrates that the HRE of this promoter is the primary target of NO. In addition, VEGF gene regulation by NO, as well as by hypoxia, is potentiated by the AP-1 element of the gene. Our study also reveals that NO and hypoxia induce an increase in HIF-1 binding activity and HIF-1 protein levels, both in the nucleus and the whole cell. These results suggest that there are common features of the NO and hypoxic pathways of VEGF induction, while in part, NO mediates gene transcription by a mechanism distinct from hypoxia. This is demonstrated by a difference in sensitivity to guanylate cyclase inhibitors and a different pattern of HIF-1 binding. These results show that there is a primary role for NO in the control of VEGF synthesis and in cell adaptations to hypoxia. (Blood. 2000;95:189-197)

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