Regulation of procollagen metabolism in the pressure-overloaded rat heart.

To determine the molecular events responsible for the disproportionate accumulation of myocardial fibrillar collagens during sustained hypertension, we examined the in vivo rate of procollagen synthesis, collagen accumulation, and intracellular procollagen degradation 1-16 wk after abdominal aortic banding in young rats. These measurements were correlated with tissue mRNA levels for type I and type III procollagen polypeptides. Banded animals developed moderate, sustained hypertension and mild left ventricular hypertrophy. Increased type III procollagen mRNA levels were detected early after banding and persisted for the entire observation period. Disproportionate collagen accumulation without histological evidence of fibrosis was noted within 1 wk after hypertension induction. Fibrillar collagen accumulation at this time point resulted not from a major increase in procollagen synthesis, but rather a marked decrease in the rate of intracellular procollagen degradation. Interstitial fibrosis, however, was observed 16 wk after banding. Type I procollagen mRNA levels were increased six-fold, but only after 16 wk of hypertension. These results correlated well with the results of in vivo procollagen synthesis experiments at 16 wk, which demonstrated a threefold increase in left ventricular procollagen biosynthesis. We conclude that pretranslational as well as posttranslational mechanisms regulate fibrillar collagen deposition in the myocardial extracellular matrix during sustained hypertension.

Retroviral v-myc infection of primary fetal liver cells: transformation of monocytes in vitro.

Oncogenes carried by retroviruses can alter the growth properties of many cell types. We examined the molecular mechanism by which a retrovirus containing one or a combination of oncogenes can transform and immortalize hematopoietic cells. Murine fetal liver cells were used as an enriched source of early hematopoietic cell progenitors; the cells were infected with a series of recombinant murine retroviruses capable of expressing the avian v-myc, v-H-ras and v-raf oncogenes. Three factor-independent cell lines were obtained: FL-ras/myc, FL-J2 (v-raf/v-myc) and FL-myc, a unique cell line generated using a single oncogene. Cytochemical, morphologic and phenotypic analyses indicated that these cell lines were of the monocyte lineage. Southern and Northern blot analyses revealed that the three cell lines had integrated viral DNA and were expressing the mRNA transcripts corresponding to these viral oncogenes. To examine the mechanism of factor independence, supernatants from these cell lines were tested for CSF-1 activity. Supernatants from FL-myc and FL-ras/myc cells were shown to contain CSF-1 activity and Northern blot analysis of the three cell lines revealed the presence of mRNA transcripts for the CSF-1 and c-fms genes. It is possible that the growth factor independence of these cell lines is related to the development of autocrine-induced proliferation.

Transforming growth factor-beta 1 in heart development.

Defined biochemical stimuli regulating neonatal ventricular myocyte (cardiomyocyte) development have not been established. Since cardiomyocytes stop proliferating during the first 3-5 days of age in the rodent, locally generated 'anti-proliferative' and/or differentiation signals can be hypothesized. The transforming growth factor-beta (TGF-beta) family of peptides are multifunctional regulators of proliferation and differentiation of many different cell types. We have determined in neonatal and maturing rat hearts that TGF-beta 1 gene expression occurs in pups of both normotensive (Wistar Kyoto, WKY) and hypertrophy-prone rats (spontaneously hypertensive, SHR). TGF-beta 1 transcript levels were readily apparent in total ventricular RNA from SHR pups within 1 day of age and elevated in 3-7 day old WKY and SHR hearts when cardiomyocyte proliferation indices are diminished. TGF-beta 1 transcript levels remain at a 'relatively' high level throughout maturation and into adulthood in both strains. Further, TGF-beta 1 transcripts were localized to cardiomyocytes of neonatal rat ventricular tissue sections by in situ hybridization. Immunoreactive TGF-beta was co-localized to the intracellular compartment of neonatal cardiomyocytes at the light and electron microscopic level. In vitro analysis using primary cultures of fetal and neonatal cardiomyocytes indicated that TGF-beta s inhibit mitogen stimulated DNA synthesis and thymidine incorporation. From these data, we propose that locally generated TGF-beta s may act as autocrine and/or paracrine regulators of cardiomyocyte proliferation and differentiation as intrinsic components of a multifaceted biochemical regulatory process governing heart development.

Insulin-like growth factor-I messenger ribonucleic acid in the developing human placenta and in term placenta of diabetics.

Fetal growth and development are dependent upon the growth and development of the placenta. Control of placental growth and development is little understood. Immunoreactive insulin-like growth factor-I and -II (IGF-I and IGF-II) have been shown to be released by human placental tissue and human placental membranes have been observed to contain specific receptors for these growth factors. Furthermore, we have demonstrated the presence of IGF-II mRNA transcripts in the developing human placenta and at gestational term in placentae of diabetics. Thus, the IGFs may have a regulatory role in the growth and development of the placenta via autocrine and/or paracrine mechanism(s) of action. In this report we demonstrate the presence of four differing size species of placental poly(A)+ RNA which specifically hybridize to an IGF-I probe originally isolated from an adult human liver cDNA library and localize IGF-I and IGF-II mRNA to syncytiotrophoblasts and fibroblasts, respectively, of the placenta by in situ hybridization. The major transcript is 7500 bases in size and the remaining three transcripts are 5000, 1100, and 900 bases in length with no apparent changes from these sizes throughout gestation and at term in diabetics. Quantification by densitometry of placental IGF-I mRNA detected by dot blot hybridization indicated that first and second trimester placentae each express more IGF-I mRNA relative to that expressed in placenta at term. These results suggest that there are developmental changes in the relative amount of IGF-I mRNA expressed in the human placenta. IGF-I is, therefore, most likely important early in gestation as a placental growth factor. This time period is critical for fetal development and growth, when embryonic induction, organogenesis, and rapid cell proliferation occur.

Coordinate gene expression during neonatal rat heart development. A possible role for the myocyte in extracellular matrix biogenesis and capillary angiogenesis.

OBJECTIVE: Neonatal heart development is a period of active extracellular matrix deposition and capillary angiogenesis which follows the cessation of ventricular myocyte proliferation. The aim was to determine whether coordinate expression of growth factors by the ventricular myocyte could function to inhibit myocyte proliferation directly as well as indirectly by paracrine stimulation of non-myocyte extracellular matrix deposition and capillary angiogenesis. METHODS: Immunohistochemistry and northern blot hybridisations were performed on ventricular samples from fetal to mature animals of the spontaneously hypertensive (SHR) and normotensive control Wistar Kyoto (WKY) strains. RESULTS: Ventricular expression of types I, III, and IV collagen genes reached their "maximum" within the first 2-3 postnatal weeks and then rapidly declined. Expression of TGF beta 3 and SPARC were found to precede and accompany the changes in extracellular matrix gene expression during this same developmental period. TGF beta 3 was immunolocalised to fetal cardiomyocytes with very limited expression in neonatal/adult non-myocytes. Associated with the neonatal expression of TGF beta variants, transcripts for the type 2 IGF receptor gradually declined over the first three postnatal weeks. Myocyte TGF beta gene expression, latent TGF beta release, and paracrine mechanisms of action could be facilitated by residual type 2 IGF receptor expression to help mediate stimulation of non-myocyte extracellular matrix synthesis and deposition. CONCLUSIONS: Expression of select growth factors, growth factor receptors, and components of the extracellular matrix appear to be highly coordinated during ventricular remodelling which occurs during neonatal heart development. A paradigm is presented which integrates the expression patterns of various myocyte derived stimuli and their postulated impact on formation of the structural components of the neonatal heart by modulation of myocyte and non-myocyte cell types.

Age-related changes in isolated rat hepatocytes. Comparison of size, morphology, binucleation, and protein content.

Hepatocytes isolated from 6-, 12-, 18-, and 30-month-old female Fischer F344 rats were examined by scanning electron microscopy. No significant change in cell size with age was observed. However, the surface morphology of the cells isolated from the older animals exhibited a significant increase in surface folds. This feature did not exceed 10% of the cell population until 12 months of age and continued to increase to 31% of the cells in 30-month-old rats. From 6 to 12 months of age, there was a significant increase in protein content of the hepatocytes. No further increase in protein content occurred during senescence. An increase in percentage of binuclear cells occurred after 24 months of age. Because ploidy and binucleation increase with increasing age, it appears that the nuclear/cytoplasmic ratio changes as a function of age.

Effects of levamisole on primary cultures of adult rat hepatocytes.

Levamisole represents one of several new compounds that exhibit immunomodulating activity. Pharmacological data have documented a relationship between liver drug metabolism of levamisole and its subsequent immunomodulating activity. To directly investigate this relationship in a controlled manner, primary cultures of adult rat hepatocytes were treated with levamisole, and ultrastructural and biochemical effects were analyzed. Ultrastructurally, levamisole did not disrupt the cellular architecture of the hepatocytes. Biochemically, levamisole stimulated alkaline phosphatase activity and elevated microsomal cytochrome P-450 content after a 48-hr incubation. High pressure liquid chromatographic analysis of levamisole metabolites produced by cultured hepatocytes suggested the formation of a hepatocyte-specific metabolite(s) that may be associated with its immunological mode of action.

Insulin-like growth factors and neonatal cardiomyocyte development: ventricular gene expression and membrane receptor variations in normotensive and hypertensive rats.

Defined factors regulating or influencing mammalian ventricular myocyte (cardiomyocyte) development are not known at this time. During early neonatal ventricular growth, cardiomyocytes begin a 'transition phase' of development toward cellular maturation (hypertrophy) that entails terminal proliferation and cellular binucleation. Insulin-like growth factor-I and -II (IGFs) are believed to play a major role in mammalian postnatal and fetal growth, possibly functioning in local environments which facilitate autocrine or paracrine tissue growth characteristics. Therefore, we examined the expression of the IGF genes and their corresponding membrane receptors in ventricles of normotensive and spontaneously hypertensive (SHR) rat pups during the first 7-14 days of age. We have determined: (1) by receptor crosslinking that neonatal ventricular membranes possess type 1 and type 2 IGF receptors; (2) by receptor binding analysis that type 1 IGF receptor concentration is elevated between days 1-7 in the SHR and shows an age-related decline in concentration and an increase in affinity in both strains; (3) by Northern blot analysis that neonatal rat ventricular tissue expresses primarily IGF-II RNA transcripts of 3.6, 2.3 and 1.7 kilobases (kb) in size, with low levels of IGF-I transcripts detected; (4) by slot-blot hybridization that SHR ventricles contain higher levels of IGF-II transcripts at 3 days of age; and (5) localized the IGF transcripts to ventricular myocytes by tissue in situ hybridization. These observations support a role for cardiomyocyte-produced IGFs that may be locally produced and act in an autocrine or paracrine fashion to modulate cardiomyocyte growth and maturation in the developing rat heart. Because both IGF receptor and IGF RNA transcript parameters differed in SHR hearts, genetically predisposed to hypertrophy, a potentially important biochemical alteration may be associated with the fetal/neonatal growth abnormalities of the developing heart in this rat strain.

Removal of surgically induced fibrous arterial plaques by argon ion laser angiosurgery using a multifiber delivery system. An experimental study in the dog.

Removal of intravascular atherosclerotic obstructions by laser irradiation has gained the attention of many investigators, but has proven to be considerably more difficult to accomplish than initially envisioned. We tested, in an animal model, an argon ion laser delivery system that permits control of (1) laser power, (2) exposure time, and (3) laser beam spot size. The study was conducted on surgically, induced focal fibrous plaques in the carotid arteries of nine dogs. Plaque removal, vessel patency, and healing were evaluated angiographically and by light and electron microscopy at intervals up to 60 days after treatment. Results showed that intravascular obstructions could be removed, healing occurred, and vessels remained patent for up to 60 days.

Role of transforming growth factor-beta 1 in regulation of hematopoiesis.

The data presented above suggest that one possible clinical use of TGF-beta would be to protect the bone marrow from the effects of myelosuppressive chemotherapeutic drugs by preventing entry or removing primitive stem cells from the cell cycle. It may also have the additional benefit of reducing the drug-induced neutrophil nadir by stimulating granulopoiesis. The availability of large quantities of recombinant TGF-beta will allow study of the pharmacokinetics with different routes of administration, dosage effects, and details of the pleiotropic effects on other cell systems. Experiments are in progress to determine whether TGF-beta will allow the delivery of higher amounts or more frequent doses of chemotherapeutic drugs and thus allow increased antitumor efficacy in tumor-bearing animals.

Biochemical characterization of neonatal cardiomyocyte development in normotensive and hypertensive rats.

Final cellular replication and functional maturation of the mammalian ventricular myocyte (cardiomyocyte) is known to occur during the first 2 post-natal weeks in rodents, yet the exact temporal sequence of events remains to be clearly established. When a genetic predisposition toward ventricular hypertrophy also manifests its influence during this time period, possible growth aberrations may occur. Using the spontaneously hypertensive rat (SHR) as an animal model, we have determined that biochemical and morphological indices of cardiomyocyte replicative deficits occur during the first 5 to 8 post-natal days relative to the normotensive control strain, Wistar Kyoto (WKY). Reduced ventricular nucleic acid (DNA and RNA) content and disproportionate RNA- and protein-DNA ratios were found in the neonatal SHR. Incorporation of tritiated thymidine into acid precipitable DNA during the first 5 postnatal days was also reduced in the SHR. Morphological evidence of reduced thymidine incorporation was determined autoradiographically at the cellular and nuclear level using isolated cells and nuclei, respectively. The results of our study indicate that SHR cardiomyocyte hyperplasia is reduced during the first 5 to 8 post-natal days when the last "window" of ventricular myocyte cellular replication occurs. Therefore, the maturing ventricle of the neonatal SHR contains fewer cardiomyocytes than its normotensive control and these cells appear to enter a hypertrophic growth phase at an accelerated rate.

Contractile activity modulates myosin heavy chain-beta expression in neonatal rat heart cells.

To determine whether spontaneous contractile activity affected the expression of myosin heavy chain isoenzymes in cultured neonatal rat heart cells, ventricular myocytes were isolated from 2-day-old rat pups by collagenase digestion and cultured for 24-96 h in the presence and absence of verapamil (10 microM), KCl (50 mM), or dihydropyridine receptor antagonists that produced contractile arrest. Inhibition of spontaneous contractile activity was associated with significant reductions in total myosin heavy chain (MHC) content and synthetic rates. Electrophoretic analysis of MHC isoenzymes indicated that MHC-beta protein rapidly disappeared from arrested cells, whereas MHC-alpha isoenzyme levels were less affected. In association with these protein changes, mRNA transcript levels for MHC-beta were markedly reduced in quiescent cells, whereas mRNA transcript levels for several other contractile protein genes were relatively less affected. Inhibition of contractile activity and MHC-beta expression were reversible upon removal of the arresting agents. Furthermore, the decrease in MHC-beta mRNA levels in arrested myocytes could be prevented by direct activation of protein kinase C with phorbol 12-myristate 13-acetate (without restoration of contractile activity). Conversely, MHC-beta mRNA levels in beating cells were reduced by treatment with staurosporine (a selective protein kinase C inhibitor). Thus contractile arrest (produced by either L-channel blockade or membrane depolarization) inhibited the accumulation of MHC-beta in cultured neonatal rat heart cells via a pretranslational mechanism. These effects may occur in response to the modulation of signaling system(s) involving mechanical "stretch" transduced via protein kinase C.

Coordinate TGF-beta receptor gene expression during rat heart development.

Transforming growth factor-beta (TGF-beta) has been implicated to participate in heart development. The receptors which transduce the signal(s) mediated by TGF-beta ligand binding have only recently been cloned. One of the most prominent effects of TGF-beta is inhibition of cell proliferation, a process that is tightly regulated during heart development. Using the developing rat ventricle as a model system, we have determined the steady state expression patterns for the Type I, II, and III TGF-beta receptors (TGF-beta Rs). Using RNA isolated from ventricular chambers on day 18 of gestation through the ninth postnatal week of age, we detected a modest increase in expression levels for Type I and Type III TGF-beta Rs. In contrast, steady state transcript levels for the Type II TGF-beta R showed a profound developmental increase from nearly undetectable levels at the fetal ages examined to high levels during the first postnatal week of age. Immunoelectron microscopic localization of Type II TGF-beta R confirmed that the 3-week-old ventricular myocyte, as well as nonmyocytes, contained immunoreactive material. Immunoreactivity was found at both the cell surface as well as intracellular compartment. Regional variations (right ventricle, left ventricle, or septum) in the expression pattern of several markers of heart development, but not the TGF-beta R's, were found in RNA obtained from 3-week-old postnatal animals. These data suggest that "downstream" effectors of TGF-beta-mediated stimulation are modulated in a developmental, regional-specific manner in the neonatal/mature myocardium by the level of bioactive TGF-beta.

Maintenance and induction of cytochrome P-450 in cultured rat hepatocytes.

Maintenance of microsomal cytochrome P-450 content by cultured rat hepatocytes has proven an elusive goal. It is reported here that exogenous heme maintains cytochrome P-450 content of cultured rat hepatocytes at high levels during the first 72 h of incubation. The maintenance studies have been expanded to demonstrate the in vitro induction of cytochrome P-450 by phenobarbital treatment. The induction of P-450 in vitro by phenobarbital required the trace element, selenium, in the presence of exogenous heme. The present findings suggest that selenium, and other trace elements, may have an essential role in the formation of holocytochrome P-450 in vitro.

Age-related changes in ploidy levels and biochemical parameters in cardiac myocytes isolated from spontaneously hypertensive rats.

Postnatal growth of the mammalian ventricular myocyte is characterized by a brief period of hyperplasia followed by an extensive period of physiological hypertrophy. Using myocytes isolated from both Wistar-Kyoto and spontaneously hypertensive rats from fetus to 10 months old, we analyzed morphological, biochemical, and ploidy changes. Fetal myocytes from both spontaneously hypertensive and Wistar-Kyoto rats were mononuclear, diploid cells. By 4 weeks, adult binucleation levels (84% binuclear) were found, and myocytes pooled from both ventricles demonstrated nuclear ploidy shifts to tetraploid levels. However, analysis of myocytes isolated from left ventricle, right ventricle, and septum showed that nuclear polyploidation was confined to the right ventricle and septum, with few polyploid nuclei detected in the left ventricle. This pattern remained relatively constant through 10 months of age, although spontaneously hypertensive myocytes showed significantly more polyploidation than Wistar-Kyoto in all regions. Biochemical analysis of isolated myocytes substantiated the nuclear ploidy changes and demonstrated elevated protein and ribonucleic acid content in left ventricular myocytes without extensive polyploidation. Since cardiac hypertrophy, both physiological and pathological, is associated primarily with the left ventricle and occurs in the absence of significant ploidy changes, these findings suggest that a unique pattern of gene regulation may be ongoing in the left ventricle myocytes that is not present in the septum and right ventricle. These variations may be essential for cellular hypertrophy under normal and pathological conditions.

Immediate postnatal rat heart development modified by abdominal aortic banding: analysis of gene expression.

Proliferative growth of the ventricular myocyte (cardiomyocyte) is primarily limited to embryonic, fetal and very early neonatal periods of heart development. In contrast, cardiomyocyte maturation, as evidenced by cellular hypertrophy, is a long-term process that can occupy the bulk of the life-span of the mature organism. As the newborn myocyte undergoes a 'transition' from proliferative to hypertrophic growth, ventricular remodeling of the non-myocyte compartment is characterized by increased extracellular matrix (ECM) formation and coronary capillary angiogenesis. A role for ventricular-derived growth factors (GFs) in these inter-related processes are examined in an animal model of altered heart development produced by neonatal aortic banding. The suprarenal abdominal aorta of five day old rat pups were banded (B), sham operated (S), or untreated (C) and ventricular tissue (left ventricular free wall and septum) obtained at 7-, 14-, and 21-days post-intervention. Using Northern blot RNA hybridizations, expression of growth factors (GFs) and/or GF-receptors (GFR's) temporally associated with heart development were evaluated. Transcript levels for TGF-beta 1, IGF-II, and their associated cell surface receptors were increased in B animals. Concomitant changes in extracellular matrix (ECM) genes (as evaluated by Collagens Type I, III, and IV) were also increased in B animals. In addition, transcript levels for the vascular morphogenesis and remodeling-related protein SPARC (Secreted Protein, Acidic and Rich in Cysteine) was also elevated in the B animals. In several instances, S animals demonstrated changes in steady state transcript levels for genes which may influence myocyte maturation during the postnatal period. This suggests that normal autocrine/paracrine growth regulatory stimuli and responses can be modified (by surgical intervention and/or abdominal aortic banding) and these perturbations in gene expression may be related to previously documented changes in myocyte cell number, vascular composition, and ventricular architecture of the banded, neonatal heart. Future studies using this model will provide an opportunity to evaluate and possibly identify the stimuli and signal transduction machinery that regulate the final phases of myocyte proliferation, stimulate capillary formation and ECM deposition, and orchestrate the transition to hypertrophic growth during heart development.

Acidic fibroblast growth factor and heart development. Role in myocyte proliferation and capillary angiogenesis.

Proliferative growth of the ventricular myocyte (cardiomyocyte) is primarily limited to fetal and early neonatal periods of development. In concert with the neonatal "transition" from proliferative to hypertrophic growth, ventricular remodeling of the nonmyocyte compartment is characterized by increased extracellular matrix synthesis/deposition and capillary angiogenesis. A role for locally generated and bioactive ventricular acidic fibroblast growth factor (aFGF) in these processes is proposed and substantiated by the following: 1) colocalization of aFGF peptide and fibroblast growth factor receptor (flg) transcripts to the developing fetal cardiomyocyte by immunohistochemistry, immunoelectron microscopy, and in situ hybridization, 2) continued localization of aFGF peptide and transcripts to the neonatal/mature cardiomyocyte, and 3) localization of flg immunoreactivity and transcripts to specific neonatal ventricular nonmuscle cell types. Specific ventricular cell types at distinct developmental stages appear to be responsive to ventricular myocyte-derived aFGF (myocytes in the fetal heart and nonmyocytes/endothelial cells in the neonatal heart). These data indicate that expression of aFGF and one of its receptors (flg) are most pronounced in the fetal to early neonatal ventricle, the presence of both suggesting an autocrine/paracrine growth regulatory function. As the animal matures, ventricular capillary angiogenesis may be facilitated by "release" of cardiomyocyte-derived fibroblast growth factors into the surrounding extracellular space/matrix functioning as a "paracrine" angiogenic stimuli. Therefore, the results of our study suggest that myocyte-derived aFGF may function to increase the fetal ventricular cardiomyocyte population in absolute number as well as to facilitate the subsequent increase in capillary angiogenesis that occurs during cardiomyocyte maturation and ventricular remodeling.

Effect of sodium butyrate on primary cultures of adult rat hepatocytes.

Sodium butyrate, at millimolar concentrations, seems to mediate or initiate multiple effects on many mammalian cells in culture. Although many transformed cell lines respond to butyrate treatment with acquisition of normal cellular characteristics, the effect of butyrate on a normal cell type, the parenchymal hepatocyte, has not been studied. Serum-free primary cultures of adult rat hepatocytes maintain many adult characteristics, yet after several days in culture a loss of adult characteristics occurs while fetal characteristics are often reexpressed. Therefore, we investigated whether butyrate treatment would improve the morphologic and biochemical characteristics of cultured hepatocytes. Exposure to 5 mM butyrate for 3 d did not affect hepatocyte viability or morphology but retarded the progressive decline in cytochrome P-450 levels and 5'-nucleotidase activity. The spontaneous increase in alkaline phosphatase activity was reduced and the induction of tyrosine aminotransferase was inhibited after 3 d in culture. The fetal liver characteristic, gamma glutamyltranspeptidase, was not affected by butyrate treatment. Results of this study suggest that butyrate represents a nontoxic compound capable of improving the maintenance of cell culture characteristics of adult rat hepatocytes.

Morphometric analysis of cardiac hypertrophy during development, maturation, and senescence in spontaneously hypertensive rats.

Hypertrophy of the mammalian heart, regardless of the initiating event, results in architectural remodeling of ventricular components that maintain structural and functional characteristics of this organ. Ventricular components that vary their morphology and morphometry in a hypertrophic state are the muscle cells, connective tissue elements, vasculature, or a combination of some or all of the above. Morphologic quantification of the progressive tissue changes occurring throughout the natural life span of the spontaneously hypertensive and normotensive Wistar-Kyoto rats has not been thoroughly documented. Using perfused-fixed tissue from both strains at 1, 6, 12, 18, and 24 months of age, we have determined the morphometric changes that occurred in the subepicardial and midwall regions of the left ventricle. Myocyte cell size, wall thickness, and arterial blood pressure were elevated in 1-month-old spontaneously hypertensive rats, reached significance by 6 months, and remained significantly greater throughout the 24 months examined. Tissue morphometry demonstrated significant tissue component volumetric differences at 6 months in the spontaneously hypertensive rat. Age-related morphometric tissue changes occurred in both strains yet were exacerbated (percent volume of myocytes) or diminished (percent volume interstitial space) in the mature and aging spontaneously hypertensive rat. Capillary density of SHR left ventricle showed a drastic decline so that 6-month-old SHR had the same density as a senescent Wistar-Kyoto. Tissue morphometry and capillary density data strongly support the hypothesis that tissue oxygenation is diminished in the spontaneously hypertensive rat, and as a result, tissue necrosis and myocyte cell death occur.