Identification and antisense inhibition of a renin-angiotensin system in transgenic cardiomyocytes.

Cardiac myocytes (AT-1 cells) derived from heart tumors of mice transgenic for an atrial natriuretic factor promoter, SV40 large T-antigen DNA transgene, demonstrate properties consistent with normal cardiac myocytes but retain the capacity to proliferate in culture. We studied the renin-angiotensin system (RAS) and related growth regulation of these cells because AT-1 cells (or transgenically similar cells) may be useful to repair injured myocardium. This study reveals two separate and distinct findings: 1) AT-1 cells proliferate or hypertrophy in response to angiotensin II (ANG II), depending on their competence to proceed through the cell cycle; and 2) AT-1 cells possess components of a RAS, and angiotensinogen antisense experiments suggest that the RAS is functional in these cells. Specifically, AT-1 cells proliferate in response to ANG II in low-serum medium but hypertrophy in response to ANG II when first treated with mitomycin C (at a concentration that inhibits DNA replication but is not cytotoxic). The ANG II-mediated proliferative and hypertrophic responses are inhibited by DuP 753. In addition, there is a significant increase in the protein-to-DNA ratio of cells, which are proliferation-inhibited in the absence of ANG II treatment (20%, P < 0.05). DuP 753 also inhibits this hypertrophy, suggesting that these cells possess a functional RAS. AT-1 cells contain mRNAs for angiotensin-converting enzyme, renin, angiotensinogen, and the AT1 receptor as determined by sequence analysis of polymerase chain reaction amplification products. Antisense oligonucleotides complementary to the angiotensinogen mRNA specifically inhibit angiotensinogen mRNA accumulation and proliferation of AT-1 cells. In summary, these cells contain a growth-regulating RAS, suggesting that such a system may play a significant role in left ventricular hypertrophy.

ACE inhibitor-mediated attenuation of mesangial cell growth. A role for endothelin.

Endothelin modulates human mesangial cell (HMC) proliferation in response to angiotensin II (Ang II). Angiotensin converting enzyme inhibitors (ACEIs) have variable effects on HMC growth depending on culture conditions. No studies, however, have investigated the effects of ACEIs on HMC production of endothelin-1 in either actively proliferating or quiescent HMCs. The present study was designed to evaluate the effects of ACEIs on HMC-associated mitogenesis, cell counts, and endothelin-1 production in the presence and absence of insulin in both quiescent and proliferating HMCs. It tests the hypothesis that ACEIs attenuate HMC growth through a reduction in HMC-associated endothelin-1 generation. The effects of four different ACEIs, an Ang II receptor antagonist, losartan, and a monoclonal antibody to endothelin-1 were evaluated. ACEIs inhibited HMC mitogenesis and cell counts in proliferative but not quiescent cells. This was due to the absence of ACE activity in HMCs and its presence in 10% fetal calf serum. Both ACEIs and losartan reduced endothelin-1 production per cell. Compared to vehicle, losartan reduced the amount of endothelin-1 in conditioned media to a greater extent than any ACEI (2.2 +/- 0.3, captopril v 1.9 +/- 0.5, quinaprilat v 3.8 +/- 0.3 delta pg/cell x 10(-3) endothelin-1, losartan; P < .05). Moreover, insulin potentiated the antimitogenic effects of both ACEIs and losartan on HMCs. Lastly, the attenuated increase of endothelin-1 in conditioned media and associated antimitogenic effect on HMCs with losartan alone was not potentiated by the addition of any ACEI to losartan. These data provide indirect evidence that Ang II production may occur in culture media when both its precursors and a sufficient amount of converting enzyme activity are present. This is predicated on the observation that HMCs lack ACE activity and that ACEIs blunt mitogenesis of proliferating HMCs. The kinetics of this reaction, as well as the mechanism of how insulin potentiates the antimitogenic effects of ACEIs, were not studied.

The use of antisense oligonucleotides to establish autocrine angiotensin growth effects in human neuroblastoma and mesangial cells.

Local renin-angiotensin systems (RAS) exist in many cell types, and angiotensin II (AII) has growth regulatory effects in some tissues. We demonstrated the presence of angiotensinogen (ANG) mRNA in cultured human mesangial cells (MC) and SHSY-5Y human neuroblastoma cells using reverse transcription and the polymerase chain reaction (RT/PCR) followed by hybridization to a human ANG-specific oligonucleotide probe. We speculated, therefore, that AII might act in an autocrine or paracrine fashion to regulate the growth of mesangial cells and neuroblastoma cells. Sense and antisense oligonucleotides were next synthesized complementary to the ANG transcription start site. Antisense but not sense oligonucleotides decreased [3H]thymidine incorporation into DNA by both MC and neuroblastoma cells. Growth of antisense oligonucleotide-treated cells was restored to control levels by the addition of AII but not by the addition of basic fibroblast growth factor. Neither oligonucleotide affected [3H]thymidine incorporation in mouse L929 cells. These data indicate that locally produced AII can act in an autocrine or paracrine fashion to alter the growth of human mesangial and neuroblastoma cells. Therefore, they suggest a role for local RAS in the pathogenesis of growth abnormalities in the cardiovascular system as well as in some forms of malignancy.

Blood pressure and nephrosclerosis in black and white men and women aged 25 to 54.

Kidney samples obtained at autopsy were evaluated for nephrosclerosis by quantitatively measuring the severity of intimal fibroplasia in the interlobular arteries and counting the number of hyalinized arterioles. Most cases were obtained from the coroner and had violent causes of death; subjects with cardiovascular diseases were excluded. Blood pressures in Health Survey data were obtained from published sources. Measures of nephrosclerosis in 365 black and white men and women in age groups 25 to 34, 35 to 44, and 45 to 54 yr were examined by three-way Analysis of Variance (ANOVA). Blacks exceeded whites in fibroplasia at all ages and in both sexes; this excess was paralleled by mean blood pressure in all comparisons. White women were not different from white men in fibroplasia; black women significantly exceeded black men at all ages. Women had lower blood pressures than men when matched for age, race, and fibroplasia; the average magnitude of this difference was 5.5 mm Hg. Since women are known to have as much hypertensive disease as men after age 50 yr, it seems possible that the arterial fibroplasia seen in young women may represent progression toward the hypertensive state, even through blood pressure does not reveal the equality of the sexes in this respect until after menopause. Hence, in women arterial fibroplasia is seen to precede a later rise in blood pressure. Arteriolar hyalinization was greater in men than in women but did not differ significantly between races. Hyalinization did not consistently follow blood pressure in comparisons between subgroups of subjects.