Kristin E. Ellison

Intimal hyperplasia after vascular injury is inhibited by antisense cdk 2 kinase oligonucleotides.

The cell cycle regulatory enzyme, cdk (cyclin-dependent kinase) 2 kinase, is activated in the rat carotid artery after balloon angioplasty injury, and may mediate smooth muscle proliferation. To test the hypothesis that inhibition of the expression of this key enzyme can inhibit intimal hyperplasia, we studied the effect of antisense phosphorothioate oligodeoxynucleotides (ODN) against cdk 2 kinase administered by intraluminal delivery using hemagglutinating virus of Japan (HVJ)-liposome-mediated transfer. The specificity of antisense cdk 2 ODN was confirmed by the observation that mRNA level of cdk 2 kinase in injured vessels was markedly diminished by the antisense ODN treatment. At 2 wk after transfection, antisense cdk 2 ODN treatment (15 microM) resulted in a significant inhibition (60%) in neointima formation, compared with sense ODN-treated and untreated vessels. Since we have previously observed that cell division cycle 2 kinase mRNA was also activated after vascular injury, we administered the combination of antisense cdc 2 and cdk 2 ODN in this study. Antisense cdc 2 ODN alone (15 microM) only reduced intimal formation by 40%. Combined antisense treatment resulted in near complete inhibition of neointima formation. To understand the mechanism of the sustained effect of a single antisense ODN administration, we examined kinetics of ODN in the vessel wall. Using phosphorothioate FITC-labeled ODN, we transfected carotid artery using the HVJ-liposome method. Fluorescence localized immediately to the medial layer, and persisted up to 2 wk after transfection. These results demonstrate that a single intraluminal administration of antisense ODN directed to cell cycle regulatory genes (e.g., cdk 2 kinase) using the HVJ method can result in a sustained inhibition of neointima formation after balloon angioplasty in rat carotid injury model.

In situ hybridization evidence for angiotensinogen messenger RNA in the rat proximal tubule. An hypothesis for the intrarenal renin angiotensin system.

We examined angiotensinogen gene expression in rat kidney by in situ hybridization histochemistry. Using a rat cRNA probe to angiotensinogen, we demonstrated angiotensinogen mRNA to be localized predominantly in the proximal renal tubule, with considerably lesser amounts in distal tubular segments and glomerular tufts. Previous studies have localized renin immunoreactivity to the juxtaglomerular cells, glomerular tufts, and proximal tubules. Such findings provide further evidence for a local tissue renin angiotensin system within the kidney which may influence regional function. Based on our data, we hypothesize that a major site of angiotensin production is the proximal tubule. We postulate that angiotensin synthesized in and/or around the proximal tubule may directly modulate tubular transport of sodium, bicarbonate, and water. In addition to the proximal tubule, the specific localization of the renin angiotensin components elsewhere in the kidney would also support the other proposed regional functions of the intrarenal system, including modulation of tubuloglomerular balance.

Evidence for direct local effect of angiotensin in vascular hypertrophy. In vivo gene transfer of angiotensin converting enzyme.

In vitro studies have demonstrated that angiotensin (Ang) II directly stimulates vascular smooth muscle cell (VSMC) growth. However, it is still unclear if Ang II exerts a direct effect on vascular hypertrophy in vivo independent of its effect on blood pressure. In vivo gene transfer provides the opportunity to assess the effects of increased activity of the vascular angiotensin system in the intact animal while avoiding an increase in circulating angiotensin or in blood pressure. Accordingly, we transfected the human angiotensin converting enzyme (ACE) vector into intact rat carotid arteries by the hemagglutinating virus of Japan-liposome method. 3 d after transfection, we detected increased ACE activity in the transfected artery. Immunohistochemistry localized immunoreactive ACE in the medial VSMC as well as in the intimal endothelial cells. The increase in vascular ACE activity was associated with a parallel increase in DNA synthesis as assessed by BrdU (bromo-deoxyuridine) index and vascular DNA content. This increase in DNA synthesis was abolished by the in vivo administration of an Ang II receptor-specific antagonist (DuP 753). Morphometry at 2 wk after transfection revealed an increase in the wall to lumen ratio of the ACE-transfected blood vessel as compared with control vector transfected vessels. This was accompanied by increases in protein and DNA contents without an increase in cell number. Local transfection of ACE vector did not result in systemic effects such as increased blood pressure, heart rate, or serum ACE activity. These morphological changes were abolished by the administration of the Ang II receptor antagonist. In this study, we used in vivo gene transfer to increase local expression of vascular angiotensin converting enzyme and provided proof that increased autocrine/paracrine angiotensin can directly cause vascular hypertrophy independent of systemic factors and hemodynamic effects. This approach has important potentials for defining the role of autocrine/paracrine substances in vascular biology and hypertension.

Identification of renin and angiotensinogen messenger RNA sequences in mouse and rat brains.

Components of the renin angiotensin system have been demonstrated in mouse and rat brains. However, local synthesis of renin has not been documented. In this study, we employed mouse submandibular gland renin complementary DNA (pDD-1D2) and rat liver angiotensinogen complementary DNA (pRang3) to examine whether renin and angiotensinogen RNA sequences exist in mouse and rat brain. Angiotensinogen messenger RNA sequences were readily demonstrable in whole rat and mouse brain using Northern blot hybridization analysis. Using large quantities (greater than 100 micrograms) of brain total RNA and the sensitive complementary RNA probe, we were able to detect low levels of renin RNA sequences in the brains of both species. The relatively low concentration of brain renin messenger RNA and high concentration of angiotensinogen messenger RNA raises several interesting questions about the distribution of these two proteins and their relative contribution to activity of the brain renin-angiotensin system. In summary, our data demonstrate the expression of both renin and angiotensinogen genes in mouse and rat brains and provide definitive evidence for an independent endogenous brain renin angiotensin system.

Androgen regulation of rat renal angiotensinogen messenger RNA expression.

Renal angiotensinogen (ang-n) mRNA concentration in the male WKY rat increases significantly during puberty. Furthermore, renal angiotensinogen mRNA level in the adult female WKY rat is considerably lower than in the male. The present study investigates the role of androgen in differential renal ang-n mRNA expression. Northern and slot blot analyses with alpha-32P labeled ang-n cDNA (pRang 3) demonstrated that castration lowered ang-n mRNA levels in the male kidney by greater than or equal to 60% compared with control, suggesting that androgen may be involved with renal ang-n gene regulation. Moreover, male WKY rats castrated as weanlings and normal adult female WKY rats each implanted with testosterone displayed significant (P less than 0.05) increases in renal ang-n mRNA levels. Our observations, taken together with previous reports that androgen influences proximal tubule morphology and the tubular expression of transport proteins (e.g., Na+/H+ antiporter), may have important physiological implications for understanding the relationship between androgen and angiotensin in the regulation of tubular function.

Mapping and Radiofrequency Catheter Ablation of the Three Types of Sustained Monomorphic Ventricular Tachycardia in Nonischemic Heart Disease

Ventricular Tachycardia Ablation, Introduction. Sustained monomorphic ventricular tachycardia (VT) associated with nonischemic cardiomyopathy (CMP) is uncommon. Optimal approaches to catheter mapping and ablation are not well characterized, but they are likely to depend on the VT mechanism. The purpose of this study was to evaluate the mechanisms of sustained monomorphic VT encountered in nonischemic CMP and to assess the feasibility, safety, and efficacy of catheter radiofrequency ablation for treatment.

Sodium regulation of angiotensinogen mRNA expression in rat kidney cortex and medulla.

Rat liver angiotensinogen cDNA (pRang 3) and mouse renin cDNA (pDD-1D2) were used to identify angiotensinogen and renin mRNA sequences in rat kidney cortex and medulla in rats on high and low salt diet. Angiotensinogen mRNA sequences were present in renal cortex and medulla in apparently equal proportions, whereas renin mRNA sequences were found primarily in renal cortex. Average relative signal of rat liver to whole kidney angiotensinogen mRNA was 100:3. Densitometric analysis of Northern blots demonstrated that renal cortical angiotensinogen mRNA concentrations increased 3.5-fold (P less than 0.001) and medulla, 1.5-fold (P less than 0.005) on low sodium compared with high sodium diet, whereas renal cortex renin mRNA levels increased 6.8-fold (P less than 0.0005). Dietary sodium did not significantly influence liver angiotensinogen mRNA levels. These findings provide evidence for sodium regulation of renal renin and angiotensinogen mRNA expressions, which supports potential existence of an intrarenally regulated RAS and suggest that different factors regulate renal and hepatic angiotensinogen.

Renin expression by vascular endothelial cells in culture.

Cultured bovine aortic endothelial cells were examined for renin activity by biochemical, immunological, and immunohistochemical techniques. When cell sonicates were incubated with renin substrate, linear generation of angiotensin I was observed (1.12 +/- 0.2 ng angiotensin I/10(6) cells per hr). The effect of pH on this activity was similar to that of bovine renal renin, and renin antibodies inhibited a large portion of the enzymatic activity. Furthermore, immunofluorescence microscopy with antirenin antisera confirmed the presence of renin within these cells. Biosynthetic radiolabeling, followed by immunoprecipitation, demonstrated de novo synthesis of a renin precursor in the endothelial cells, which was processed to a more mature protein. Thus, bovine aortic endothelial cells in culture contain and biosynthesize renin, a key component of the renin-angiotensin system. The expression of renin activity by endothelium may contribute to the local regulation of vascular tone.

Identification and Ablation of Macroreentrant Ventricular Tachycardia with the CARTO Electroanatomical Mapping System

Monomorphic ventricular tachycardias associated with regions of scar are most commonly due to reentry. Catheter based techniques have recently been described for mapping of reentry circuits. Fluoroscopic methods have obvious limitations when attempting to map large ventricular reentry circuit and localize target‐sites of radiofrequency ablation. Three‐dimensional right ventricular endocardial mapping was performed in a 38‐year‐old patient with ventricular tachycardia 28 years after surgical correction of tetralogy of Fallot by using the CARTO electroanatomical system. The map of electrogram voltage showed low amplitude electrograms over the anterior wall of the right ventricle extending into the right ventricular outflow tract, consistent with the location of the ventriculotomy scar. Recording of local activation time was combined with entrainment mapping to define the macroreentrant circuit during ventricular tachycardia. Since the activation propagated through a broad path around the right ventriculotomy scar, ablation was performed by creating a line of block, which was facilitated by tagging of the lesion sites on the endocardial activation map. Large ventricular reentry circuits can be identified and interrupted by creation of a line of block to interrupt a broad path. A practical approach to mapping combining analysis of electrogram voltage, activation sequence, and entrainment is presented.

Catheter Ablation for Hemodynamically Unstable Monomorphic Ventricular Tachycardia

Ablation of Unstable Ventricular Tachycardia. Introduction: Hemodynamic collapse precludes extensive catheter mapping to identify focal target regions in many patients with ventricular tachycardia (VT) associated with heart disease. This study tested the feasibility of catheter ablation of poorly tolerated VTs by targeting a region identified during sinus rhythm.