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Biomedical subjects

Michael J Katovich

Publications and source records attributed to Michael J Katovich.

6 recordsLinked to original sources
PubMed2003

Efficient large-scale production and concentration of HIV-1-based lentiviral vectors for use in vivo.

The aim of this study was to develop an efficient method for packaging and concentrating lentiviral vectors that consistently yields high-titer virus on a scale suitable for in vivo applications. Transient cotransfection of 293T packaging cells with DNA plasmids encoding lentiviral vector components was optimized using SuperFect, an activated dendrimer-based transfection reagent. The use of SuperFect allowed reproducible and efficient production of high-titer lentiviral vector at concentrations greater than 1 x 10(7) transducing units per ml (TU/ml) and required less than one-third of the total amount of DNA used in traditional calcium phosphate transfection methods. Viral titers were further increased using a novel concentration protocol that yielded an average final titer of 1.4 x 10(10) TU/ml. Lentiviruses produced using these methods exhibited efficient transduction of central nervous system and peripheral tissues in vivo. The method is reproducible and can be scaled up to facilitate the use of these vectors in animal studies.

Alkaline Phosphatase↗
PubMed2003

Endothelial-independent prevention of high blood pressure in L-NAME-treated rats by angiotensin II type I receptor antisense gene therapy.

It has previously been established that a single systemic administration of retroviral vector containing angiotensin II type I receptor antisense (AT(1)R-AS) in the neonatal spontaneously hypertensive rat (SHR) prevents development of hypertension, and in addition cardiac hypertrophy and endothelial dysfunction. However, these studies could not determine whether the effects of AT(1)R-AS on high blood pressure (BP) and endothelial function were independent. Angiotensin receptor blockers have been shown to reduce BP in the L-NAME (N (omega)-nitro-L-arginine methyl ester hydrochloride)-induced rat model of hypertension. Our objective in the present study was to use the L-NAME model of hypertension to determine whether AT(1)R-AS treatment would lower high BP and attenuate cardiac hypertrophy under conditions of permanent endothelial damage. A single bolus of LNSV-AT(1)R-AS viral particles in neonatal Wistar-Kyoto (WKY) rats was without affect on basal BP. Efficacy of the transgene incorporation was assessed by observing a significant reduction in angiotensin-induced dipsogenic response in the AT(1)R-AS-treated animals. Introduction of L-NAME in the drinking water for 10 weeks resulted in the establishment of hypertension only in the WKY rats treated with vector alone. These hypertensive (BP, 179 +/- 4 mmHg) animals showed a 17 % increase in heart weight/body weight ratio and a 60 % reduction in ACh-induced vasorelaxation in phenylephrine-preconstricted arteries. The L-NAME-induced high BP and cardiac hypertrophy were attenuated in rats expressing AT(1)R-AS. However, endothelial dysfunction could not be prevented with the antisense therapy. These observations demonstrate that attenuation of endothelial dysfunction is not a prerequisite for the antihypertensive effects of AT(1)R-AS treatment.

Animals↗
PubMed2002

Hypertension-linked decrease in the expression of brain gamma-adducin.

Gene profiling data coupled with adducin polymorphism studies led us to hypothesize that decreased expression of this cytosolic protein in the brain could be a key event in the central control of hypertension. Thus, our objectives in the present study were to (1) determine which adducin subunit gene demonstrates altered expression in the hypothalamus and brainstem (two cardioregulatory-relevant brain areas) in two genetic strains of hypertensive rats and (2) analyze the role of adducins in neurotransmission at the cellular level. All three adducin subunits (alpha, beta, and gamma) were present in the hypothalamus and brainstem of Wistar Kyoto (WKY) and spontaneously hypertensive (SH) rats. However, only the gamma-adducin subunit expression was 40% to 60% lower in the SH rat compared with WKY rat. A similar decrease in gamma-adducin expression was observed in the hypothalamus and brainstem of the renin transgenic rat compared with its normotensive control. Losartan treatment of the SH rat failed to normalize gamma-adducin gene expression. A hypertension-linked decrease of gamma-adducin was confirmed by demonstrating a decrease in gamma-adducin expression in hypothalamic/brainstem neuronal cultures from prehypertensive SH rats. Neuronal firing rate was evaluated to analyze the role of this protein in neurotransmission. Perfusion of a gamma-adducin-specific antibody caused a 2-fold increase in the neuronal firing rate, an effect similar to that observed with angiotensin II. Finally, we observed that preincubation of neuronal cultures for 8 hours with 100 nmol/L angiotensin II caused a 60% decrease in endogenous gamma-adducin and was associated with a 2-fold increase in basal firing rate. These observations support our hypothesis that a decrease in gamma-adducin expression in cardioregulatory-relevant brain areas is linked to hypertension possibly by regulating the release of neurotransmitters.

Action Potentials↗
PubMed2002

Genetic targeting of the renin-angiotensin system for long-term control of hypertension.

Although traditional approaches are effective for the treatment and control of hypertension, they have not succeeded in curing the disease, and have therefore reached a plateau. As a result of the completion of the Human Genome Project and the continuous advancement in gene delivery systems, it is now possible to investigate genetic means for the treatment and possible cure for hypertension. In this review we discuss the potential of genetic targeting of the renin-angiotensin system for the treatment of hypertension. We provide examples of various approaches that have used antisense technology with a high degree of success. We focus on our own research, which targets the use of antisense of the angiotensin type I receptor in various models of hypertension. Finally, we discuss the future of antisense technology in the treatment of human hypertension.

Blood Pressure↗
PubMed2002

Blood pressure-independent attenuation of cardiac hypertrophy by AT(1)R-AS gene therapy.

Our studies have established that a single intracardiac administration of the retroviral vector containing angiotensin II type I receptor antisense gene causes prolonged antihypertensive actions in the spontaneously hypertensive rat. These results suggest that antisense gene therapy is a conceptually valid strategy for the control of hypertension at the genetic level. To evaluate whether attenuation of the pathophysiological aspects of hypertension are dependent on the blood pressure lowering actions of antisense gene therapy, we chose the renin transgenic rat as a hypertensive animal model and cardiac hypertrophy as the hypertension-associated pathophysiology. A single intracardiac administration of the retroviral vector containing angiotensin II type I receptor antisense in the neonatal rat resulted in long-term expression of the antisense transgene in various cardiovascular-relevant tissues, including the heart. This expression was associated with a significant attenuation of cardiac hypertrophy despite its failure to normalize high blood pressure. Developmental studies indicated that cardiac hypertrophy was evident as early as 16 days of age in viral vector-treated control transgenic rats, despite these animals exhibiting normal blood pressure. These observations demonstrate that, in the renin-transgenic rat, the onset of cardiac hypertrophy occurs during development and is prevented without normalization of high blood pressure. Collectively, these results provide further proof of the concept and indicate that antisense gene therapy could successfully target the local tissues' renin-angiotensin system to produce beneficial cardiovascular outcomes.

Actins↗