RAAV-mediated Decorin Gene Delivery Reverses Cardiac Fibrosis In Spontaneously Hypertensive Rats

Background and objective: Decorin (DCN), the small proteoglycan, is one memberof extracellular matrix (ECM) in the interstitial tissue and also characterized as a naturallyoccurring inhibitor that antagonizes transforming-growth factor-beta (TGF-β) which hasbeen regarded as the most important fibrotic cytokine. It has been proved that DCN canbind and neutralize extracellular TGF-βso as to inhibit organic fibrosis causing by theoverexpression of TGF-β. Thus, we conclude that DCN delivery may have therapeuticpotential for hypertensive disease. Recombinant adeno-associated virus (rAAV) has beenshown high efficiency of infecting both dividing and nondividing cells and tissues, rAAVmediated gene infection leads to stable, long-term expression without obvious immuneresponse and other adverse effects. These properties of rAAV vectors indicate that theymay constitute a powerful tool for gone therapy of chronic diseases. In the current study, weinvestigated the potential favorable effects and mechanisms of administering a recombinantadeno-associated virus (rAAV) vector expressing the DCN cDNA to reverse cardiacfibrosis in spontaneously hypertensive rats (SHR) at a dose of 1×10¹¹ p.f.u, via tail vein injection.Methods: We delivered human DCN cDNA in rAAV. The rAAV virons carryingDCN and GFP (as control) were packed by co-transfections in 293 cells and tittered by dotblots respectively. A single dose of rAAV-GFP or rAAV-DCN (about 1×10¹¹ virionparticles per rat in 1 ml of saline solution) via the tail vein into the adult SHRs was injectedrespectively. Rat' blood pressure was checked every two week and 24hs urine wereharvested. All SHRs were sacrificed 16 weeks after gene delivery and measuredcardiaovascular functional indexes, including the left ventricle end-systolicpressure(LVESP) and the left ventricle end-diastolic pressure (LVEDP) and themaximal/minimum rate of LVP (±LV dp/dtmax). DCN expression in various organs ofSHRs was assessed by Western blots, reverse transcription-polymerase chain reaction(RT-PCR) and enzyme-linked immunospecific assay (ELISA). The microalbumin level inurine was analyzed by ELISA. Renal injury and the cardiovascular remodeling wereevaluated by collagen analysis. The expression of TGF-β,α-SMA, and the levels ofphosphorylation of Smad2. p38 MAPK were measured by Western blots.Results: (1) After rAAV-mediated DCN gene administration, there were significantincreases in LVESP and +dp/dt_max, but decreases in LVEDP and enhancement in -dp/dt_maxas compared with controls. These data indicate an improvement in both systolic anddiastolic functions after rAAV-DCN treatment; (2) The result of Western blot, ELISA andthe reverse transcription-polymerase chain reaction (RT-PCR) showed that abundant DCNprotein and mRNA were expressed in heart, liver and kidney in rAAV-DCN treated SHRs;(3)Collagen analysis identified that DCN gene delivery reduced size of cardiomyocytes anddeposit of collagen in heart; (4) Urine microalbumin levels were significantly decresed indifferent time points after DCN gene delivery as compared with controls: (5) Collagenanalysis of kidney showed that compared with controls, DCN gene delivery significantlyimproved the deposition of collagen, attenuated glomerular sclerosis; (6) Results showedthat rAAV-DCN treatment significantly decreasedα-SMA expression in heart, consistent with its ability to inhibit ECM accumulation in heart, indicating rAAV-DCN treatmentinhibited differentiation of fibroblasts in heart in hypertensive rats; (7) Western blot showedthat rAAV-DCN treatment significantly attenuated TGF-β, p-smad2, p38MAPK andSmad6 expression in heart.Conclusions: Based on these results, we concluded that rAAV mediated DCN genedelivery resulted in a stable and long term expression of human DCN in SHRs andalleviated cardiac as well as renal fibrosis in SHRs. Thus, it can be made a conclusion thatrAAV mediated DCN gene can be an excellent gene target for cardiac fibrosis resultingfrom hypertension.