Recombinant adeno-associated virus-mediated kallikrein gene therapy reduces hypertension and attenuates its cardiovascular injuries

Abstract

Gene therapy of hypertension requires long-term expression of a therapeutic gene to achieve stable reduction of blood pressure. Human tissue kallikrein (HK) cleaves kininogen to produce a potent vasoactive peptide kinin, which plays an important role in the regulation of the cardiovascular and renal functions. In the present study, we have delivered human kallikrein cDNA with an rAAV vector to explore the potential therapeutic effects of kallikrein on hypertension and related secondary complications. A single tail vein injection of the rAAV-HK vector into the adult spontaneously hypertensive rats resulted in a significant reduction (12.072.55 mmHg, Po0.05, n ¼ 6, ANOVA) of the systolic blood pressure from 2 weeks after vector injection, when compared with the control rAAV-lacZ vector-injected rats. Weekly blood pressure monitoring showed stable hypertension-reduction effect throughout the course of the 20-week experiments. In addition, total urine microalbumin contents decreased as a result of rAAV-HK treatment. Histological analysis of various tissues showed remarkable amelioration of cardiovascular hypertrophy, renal injury and collagen depositions in the rAAV-treated group. Finally, persistent expression of the transgene product HK was confirmed by the enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction. We conclude that rAAV-mediated HK delivery rendered a long-term and stable reduction of hypertension and protected against renal injury, cardiac remodeling in the spontaneously hypertensive rat model. Further studies are warranted for the development of a gene therapy strategy for human hypertension.

Long-term modifications of blood pressure in normotensive and spontaneously hypertensive rats by gene delivery of rAAV-mediated cytochrome P450 arachidonic acid hydroxylase

“…In order to further investigate the relationship of CYP4A expression level and blood pressure in rats, the present study used recombinant adeno-associated virus (rAAV) as vector to overexpress 4A1 or to block the expression of CYP4A by injection of either rAAV·CYP4A1 or anti4A1, respectively, via the sublingual vein. The rAAV vector system has several unique advantages for gene transfer over other viral vectors (such as plasmid and adenoviral vectors), including a high efficiency of infection, minimal induction of host immune and inflammatory response and driving genes to express in vivo and in vitro in long-term and stably [19,32]. To our surprise, both Northern blots and Western blots demonstrated that after rAAV injection, CYP4A1 was preferentially expressed in the kidney in long-term, with only low level of expression in the liver and poorly in all the other organs.…”

Arachidonic Acid Epoxygenase Metabolites Stimulate Endothelial Cell Growth and Angiogenesis via Mitogen-Activated Protein Kinase and Phosphatidylinositol 3-Kinase/Akt Signaling Pathways

“…These epoxygenase cDNAs or GFP were subcloned into the rAAV vector pXXUF 1 downstream of the cytomegalovirus promoter and rAAV-CYP102 F87V, rAAV-CYP2C11-CYPOR, rAAV-CYP2J2, and rAAV-GFP were packed in human 293 cells (American Type Culture Collection, Manassas, VA) and purified by a single-step gravity-flow column purification method as described previously (Xiao et al, 1996;Xiao et al, 1998;Wang et al, 2003b). Purified rAAV viruses were titered using a dot blot method (Wang et al, 2004).…”

Recombinant adeno-associated virus-mediated human kallikrein gene therapy protects against hypertensive target organ injuries through inhibiting cell apoptosis

“…Animal treatment and gene delivery were described previously [6] . Twelve three-month-old male SHRs weighing 250-280 g were randomly divided into two groups (six rats in each group).…”

“…An 860-bp HK fragment (NotI/NotI) containing an open reading frame was subcloned into pXXUF 1 downstream of CMV to establish the pUF1-HK plasmid. The rAAV·HK and rAAV·LacZ viruses were produced as described previously [6] . The titer of rAAV-HK and rAAV-lacZ was determined by dot blot hybridization.…”

“…Our previous studies showed that overexpression of human tissue kallikrein (HK) mediated by rAAV caused a long-term decrease in blood pressure in spontaneous hypertensive rats (SHRs) and high salt-induced hypertensive rats and lessened injuries to the heart, aorta and kidney [6][7][8] . We hypothesized that in addition to its blood pressure-lowering effects, KKS could prevent end-organ injury by inhibiting apoptosis.…”