Kuei-Fu Lin

Prolonged Reduction of High Blood Pressure With Human Nitric Oxide Synthase Gene Delivery

Endothelium-derived nitric oxide (NO) in peripheral vessels has been shown to modulate vascular resistance and blood pressure. We explored the effect of a continuous supply of human endothelial NO synthase (eNOS) on the blood pressure of spontaneously hypertensive rats (SHR) by somatic gene delivery. A DNA construct containing the human eNOS gene fused to the cytomegalovirus promoter/enhancer was injected into SHR through the tail vein. A single injection of the naked eNOS plasmid DNA caused a significant reduction of systemic blood pressure for 5 to 6 weeks in SHR, and the effect continued for up to 10 to 12 weeks after a second injection. The differences were significant from 2 to 12 weeks postinjections (n=6, P<.01). In a separate experiment, L-arginine, the substrate of eNOS, was supplied in drinking water at a concentration of 7.5 g/L for 11 weeks after eNOS gene delivery. A maximal blood pressure reduction of 21 mm Hg in SHR was observed with eNOS DNA compared with that of control SHR injected with vector DNA (181.9+/-1.46 versus 202.7+/-2.79 mm Hg, mean+/-SEM, n=6, P<.01). Human eNOS gene delivery induces significant increases in urinary and aortic cGMP levels and urinary and serum nitrite/nitrate content (P<.05), while no significant differences in body weight, heart rate, water intake, food consumption, or urine excretion were observed. These results indicate that somatic delivery of the human eNOS gene induces a prolonged reduction of high blood pressure and raises the potential of using eNOS gene therapy for hypertension and cardiovascular diseases.

Human Endothelial Nitric Oxide Synthase Gene Delivery Promotes Angiogenesis in a Rat Model of Hindlimb Ischemia

Objective—Endothelium-derived NO has been shown to mediate the mitogenic effect of vascular endothelial growth factor on cultured microvascular endothelium. To evaluate the role of endothelial NO synthase (eNOS) in angiogenesis in the ischemic hindlimb, we engineered an adenovirus containing human eNOS cDNA. Methods and Results—After gene transfer, expression of eNOS in cultured cells was detected by increased intracellular cGMP and nitrate/nitrite levels and NO synthase activity. Adenovirus containing either the eNOS or luciferase gene was injected into the adductor muscle of rat hindlimbs immediately after femoral artery removal. Human eNOS protein was detected throughout the course of the experiment by immunostaining. Significant increases in blood perfusion were monitored by laser Doppler imaging from 2 to 4 weeks after gene delivery in the ischemic hindlimb of rats receiving eNOS compared with control rats receiving the reporter gene. An increase in regional blood flow was also detected after eNOS gene transfer by a fluorescent microsphere assay. eNOS gene delivery in the ischemic hindlimb resulted in significant increases in intracellular cGMP levels and in capillary density identified by anti–CD-31 immunostaining. Angiogenesis was further confirmed in mice after eNOS gene transfer by increased hemoglobin content in Matrigel implants. Conclusions—Taken together, these results indicate that eNOS enhances angiogenesis and raises the potential of eNOS gene transfer for modulation of vascular insufficiency.

Atrial Natriuretic Peptide Gene Delivery Reduces Stroke-Induced Mortality Rate in Dahl Salt-Sensitive Rats

Atrial natriuretic peptide (ANP) is a powerful hormone with hypotensive, natriuretic, diuretic, and many other beneficial effects. Direct infusion of ANP in therapeutics has limited success because of its short half-life in the circulation. Our previous studies have shown that ANP gene delivery attenuates hypertension, cardiac hypertrophy, and renal injury in Dahl salt-sensitive (Dahl-SS) rats. To investigate the potential therapeutic value of ANP gene delivery on salt-induced stroke and cerebrovascular disorders, an adenovirus harboring the human ANP gene (Ad.RSV-cANP) was injected into Dahl-SS rats on a high salt diet. A single intravenous injection of the ANP gene caused a significant reduction of blood pressure that lasted for more than 3 weeks. A maximal blood pressure reduction of 28 mm Hg was observed 2 weeks after gene delivery as compared with that of control rats injected with adenovirus harboring the LacZ gene under the control of the Rous sarcoma virus promoter (Ad.RSV-LacZ). Immunoreactive human ANP can be detected in the heart, lung, kidney, and brain of rats after gene delivery. The stroke mortality rate of Dahl-SS rats was significantly decreased (from 54% to 17% at 3 weeks and from 70% to 50% at 4 weeks after ANP gene delivery as compared with rats injected with control virus). ANP gene delivery also significantly attenuates salt-induced aortic hypertrophy as evidenced by reduced thickness of the aortic wall. This is the first study to demonstrate the potential of ANP gene delivery in reducing the mortality rate caused by cerebrovascular disorders and stroke. Successful application of this technology may have potential value in treating individuals with a high risk of stroke.

Adenovirus-Mediated Human Tissue Kallikrein Gene Delivery Inhibits Neointima Formation Induced by Interruption of Blood Flow in Mice

Tissue kallikrein cleaves kininogen to produce vasoactive kinin peptides. Binding of kinins to bradykinin B(2) receptors on vascular endothelial cells stimulates the release of nitric oxide and prostacyclin, thus activating the cGMP and cAMP pathways. In this study, we evaluated the effects of adenovirus-mediated human tissue kallikrein gene (Ad.CMV-cHK) delivery in a mouse model of arterial remodeling induced by permanent alteration in shear stress conditions. Mice underwent ligature of the left common carotid artery and were injected intravenously with saline or 1.8 x 10(9) plaque-forming units of Ad.CMV-cHK or control virus (Ad.CMV-LacZ). Fourteen days after surgery, morphometric analysis revealed that Ad. CMV-cHK reduced neointima formation by 52% (P<0.05) compared with Ad. CMV-LacZ. Expression of human tissue kallikrein (HK) mRNA was detected in mouse carotid artery, aorta, kidney, heart, and liver, and recombinant HK was present in the urine and plasma of mice receiving HK gene. Kallikrein gene transfer resulted in increases in urinary kinin, cGMP, and cAMP levels. The protective action of Ad. CMV-cHK on neointima formation was significantly reduced (P<0.05) in mice with knockout of the kinin B(2) receptor gene compared with wild-type control mice (J129Sv mice). In contrast, the effect of Ad. CMV-cHK was amplified (P<0.05) in transgenic mice overexpressing human B(2) receptor compared with wild-type control mice (c57/Bl6 mice). Thus, the inhibitory effect of recombinant kallikrein on structural alterations caused by the interruption of blood flow appears to be mediated by the B(2) receptor. These results provide new insight into the role of the tissue kallikrein-kinin system in vascular remodeling and suggest the application of HK gene therapy to treat restenosis and atherosclerosis.

Human tissue kallikrein attenuates hypertension and secretes into circulation and urine after intramuscular gene delivery in hypertensive rats

Systemic delivery of the human tissue kallikrein transgene has been shown to markedly delay the increase of blood pressure in hypertensive rat models. To demonstrate potential hypotensive effects of kallikrein via local delivery, adenovirus carrying the human tissue kallikrein gene was inoculated into quadriceps of spontaneously hypertensive rats (SHR). A single intramuscular injection of the kallikrein gene caused a significant delay of blood pressure increase for 5 weeks. The expression of human tissue kallikrein and its mRNA was identified solely in injected muscle. Immunoreactive human tissue kallikrein was detected in the muscle as well as in the circulation and urine of adult and newborn rats. Urinary kinin and cGMP levels increased significantly in rats receiving kallikrein gene delivery as compared with rats receiving control virus containing the LacZ gene. The detection of human tissue kallikrein in rat urine after local gene delivery into the muscle provides direct evidence that circulatory kallikrein can be secreted into the urine. These findings indicated that a continuous supply of human tissue kallikrein in the circulation is sufficient to reduce blood pressure and kallikrein gene delivery via the intramuscular route may have significant implications in therapeutic applications.