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Notes: Tissue ischemia is a common occurrence in many disease processes including chronic wounds, stroke, solid tumors, and myocardial infarction. The application of gene delivery for healing of wounds has demonstrated increasing therapeutic promises in animal models. Adenoviral vectors have been successfully used for gene delivery to the ischemic wound. However, these vectors typically demonstrate short, transient transgene expression while eliciting significant cytotoxic immune response. Adeno-associated viral vectors (AAV) do not have those limitations; however, scant information is available about their transfection efficiency under low-oxygen tension. The goal of this study was to compare AAV vector with adenoviral vector in terms of relative efficiency of gene delivery and cytotoxic immune response in ischemic wounds. Reporter constructs Ad5-LacZ and AAV-LacZ (108 pfu/wound) were injected onto the dermis of rabbit ear prior to creation of ischemic wounds. Wounds were harvested at postoperative day 10. Frozen sections of the wounds were fixed in cold acetone and stained with an in situ β-gal staining kit. Intense expression of β-gal was observed with both vectors; however, transduction rates with AAV vector was approximately 10-fold lower than Adenovirus. Unlike Adenovirus, no noticeable inflammatory cell infiltration was observed with AAV injection. Even when the dosage of AAV was increased to 109 pfu/wound inflammatory cell infiltration remained negligible. Thus our data indicates that both AAV and adenoviral vectors are suitable to use in gene-therapy experiments in ischemic tissues. The particular advantage of AAV is the ability to transfect with higher doses while at lower dose maximal transfection rate seems to be more with Adenovirus.

Notes: Accumulating data strongly suggests that under hypoxic conditions the adaptive physiological response of the cells involves co-operation between oxygen sensing and growth factor signals that cause HIF-1 mediated gene expression. The HIF-1 has been identified as a central and critical molecule in oxygen sensing and possibly a master switch. Of the many cytokines that have been shown to modulate collagen production, TGFβ1 appears to be crucial as it can sustain stimulation of collagen production as well as autoinduction of its own synthesis. Several genes that are known to be hypoxia inducible are also up-regulated by TGFβs, and the promoter of TGFβ3, a member of the family, has a hypoxia-response element (HRE). Recent reports indicate that HIF-1α physically interacts with Smad3 and that the TGFβ and hypoxia signaling pathways synergize at the transcriptional level to regulate gene expression. To determine if this interaction upregulates gene expression through the HRE element, we have co-transfected cultured human dermal fibroblasts with the mammalian expression plasmid for HIF-1α(pCEP1-HIF-1α) with a reporter construct 5HRE-luc, containing a concatemer of five copies of HRE derived from human vascular endothelial growth factor (VEGF), a minimal cytomegalovirus (CMV) promoter and a reporter gene,luciferase.). When treated with 2 ng/ml of TGFβ1 protein, co- transfected hypoxic aged and young human dermal fibroblasts showed significant synergistic upregulation of the reporter gene expression. To further confirm the TGFβ-HIF-1α interaction we have created a TGFβ1 -RNAi construct by subcloning a 19-nucleotide sequence derived from rabbit in a mammalian expression vector (p-SUPER) that directs the synthesis of small interfering RNAs (siRNAs). Hypoxic rabbit cultured dermal fibroblasts co-transfected with TGFβ1-RNAi and 5HRE-luc showed down-regulation of reporter gene expression.As a prerequisite to understanding the biology of physiological and pathological ischemic tissue repair process it is important to delineate the molecular basis of regulation of collagen gene expression in fibroblasts. Each step in the TGFβ signaling cascade is a potential target for highly specific therapy and information about how hypoxic low-oxygen microenvironments affect the TGFβ cascade could be useful to develop novel therapeutic strategies that will involve agonist and antagonist agents that directly interfere with these steps.