Abstract
The objective of this study was to investigate the effect of the PR39 recombinant adeno-associated virus (AAV) controlled by the hypoxia-responsive element (HRE) on gene therapy of ischemic heart disease. The minimal HRE was artificially synthesized and the AAV vector controlled by HRE was introduced with NT4-TAT-His-PR39 to investigate the expression of AAV-PR39 in hypoxic vascular endothelial cells (VEC) of human umbilical vein (CRL-1730 cell line) and the angiogenesis-promoting effect in pigs with acute myocardial infraction (AMI). The minimal HRE/CMV was designed and artificially synthesized using the PCR method and cloned with the T vector cloning method. The pSS-HRE-CMV-NT4-6His-PR39-PolyA-AAV plasmid was constructed. Using the calcium phosphate precipitation method, HEK-293 cells were co-transfected with three plasmids to produce the recombinant virus. An equal volume of pSS-HRE-CMV-NT4-6His-PR39-PolyAAAV and enterovirus (EV, blank virus) was transfected into CRL-1730 cell lines, respectively. The immunohistochemical method was used to assay the expression of 6xHis in CRL-1730 cell lines and the expression of PR39 under hypoxia. Eighteen AMI miniature pigs were randomized into the experimental group (HRE-AAV-PR39 group), control group 1 (physical saline group) and control group 2 (EV group). The area of ischemia was assessed with conventional MRI and myocardium perfusion MRI. Pigs were sacrificed at preset time-points to obtain samples of ischemic myocardium. Morphological and pathological data were collected. According to data in the literature and databases, the minimal HRE was designed and synthesized with the PCR method. A large number of HREs were connected to modified pSSHGAAV (pSSV9int-/XbaI) vector followed by insertion of the NT4-6His-PR39 gene segment and, thus, the recombinant plasmid pSS-HRE-CMV-NT4-6His-PR39-PolyA-AAV was successfully constructed. The expression of 6xHis in CRL-1730 cells under the regulation of HRE was assayed using the immunohistochemical method and results showed that the expression was positive in the experimental group. Myocardium perfusion MRI displayed that the infracted area significantly decreased under the action of pSS-HRE-CMV-NT4-PR39-PolyA-AAV. The artificial minimal HRE in CRL-1730 cells effectively and rapidly regulates the expression of the downstream gene NT4-TAT-His-PR39 of the CMV promoter. Recombinant pSS-HRE-CMV-NT4-PR39-Poly-AAAV promotes neoangiogenesis in the ischemic area, reduces the area of infarction and improves heart function.
Highlights
The hypoxia-responsive element (HRE) is the minimal indirect cis-regulatory element transactivated by hypoxia-inducible factor (HIF)
Data from over 70 genes suggest that endogenous HREs are composite regulatory elements comprising the conserved HIF-binding site (HBS) with an A/GCGTG core sequence and a highly variable flanking sequence
Digital microphotographs showed that the average OD value for positive HIF-1α was 5469±395.7 in the experimental group and 1686±423.2 in the control group (P
Summary
The hypoxia-responsive element (HRE) is the minimal indirect cis-regulatory element transactivated by hypoxia-inducible factor (HIF). Data from over 70 genes suggest that endogenous HREs are composite regulatory elements comprising the conserved HIF-binding site (HBS) with an A/GCGTG core sequence and a highly variable flanking sequence. A single HBS is essential but not sufficient for activation by hypoxia. Binding sites of transcription factors provided by the flanking sequence are not necessary for hypoxia regulation but are required to amplify the hypoxic response or make the HRE tissue-specific [1]. We firstly synthesized the minimal HRE and introduced NT4-TAT-His-PR39 to HRE-regulated adeno-associated virus (AAV) to investigate the expression in hypoxic vascular endothelial cells (VEC) of human umbilical vein (CRL-1730 cell lines) and the angiogenesis-promoting effect on myocardial cells in pigs with acute myocardial infarction (AMI)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
