Filiberto Giacomelli

Defective Oxidative Metabolism of Heart Mitochondria from Genetically Diabetic Mice

Long chain saturated β-hydroxy fatty acid content and oxidative metabolism were studied in hearts of diabetic mice (C57BL/KsJ db/db) with a progressive cardiomy-opathy at intervals of 7, 10, 16, and 26 wk of age. Total β-hydroxy fatty acid (BHFA) content increases progressively with age in diabetic hearts with a mean value of 143.5 nmoi/g dry wt as compared with a mean of 59.6 nmol/g dry wt in control hearts. There was also a redistribution of BHFA in myocardium of diabetic mice when compared with controls, with a relative decrease in β-hydroxymyristate and an increase of β-hy-droxypalmitate. Oxidative phosphorylation studies using isolated mitochondria from diabetic mice demonstrated depressed state 3 oxidation rates with both palmityl carnitine and pyruvate as substrates. While mitochondrial NADH-oxidase activity was not statistically different from that of controls, there was a significant decrease in mitochondrial total NAD + NADH content in diabetic hearts. In addition, treatment of my-ocardfal tissue with lanthanum demonstrated an abnormal permeability of sarcolemmal, intercalated disc as well as mitochondrial membranes in myocytes of diabetic mice. The data indicate that deficiencies in total NAD + NADH content can account for the depressed state 3 oxidation of palmitylcarnitine and pyruvate in diabetic mice that in turn may explain the abnormal accumulation of BFHA. The latter could play a role in altering the permeability of cardiac cell membranes.

ISolation and characterization of cerebral resistance vessel endothelium in culture

Organ-derived endothelia have been shown to exhibit distinct patterns of morphology and growth responsiveness in vitro. This report describes the development, cloning and establishment of long-term serial cultures of rat vascular endothelial cells derived from cerebrocortical resistance vessels (small arteries and arterioles). Modification of our previous published technique for establishing resistance vessel-derived smooth muscle cells (RV-SMC) resulted in enhanced levels of endothelial outgrowth from collagenase-treated microvessel fragments. Although primary culture growth consisted predominantly of SMC, subsequent subcultivation of these cultures revealed the presence of distinct endothelial cell clusters within the SMC monolayer. Serial cloning of these isolates resulted in a homogeneous population of cells with the characteristic endothelial cobblestone growth pattern and positive immunofluorescence for factor VIII-related antigen. Previously established RV-SMC frozen stocks provided an additional source for obtaining resistance vessel endothelial cells. This was made possible by the slow proliferation rate of early-passage RV-SMC and their inability to withstand freezing procedures. Endothelial cells from both preparations were identical and designated resistance vessel derived endothelial cells RV-EC. Upon long-term cultivation (> P15), confluent RV-EC cultures expressed spontaneous multicellular cord development that stained positive for factor VIII-related antigen. Cell growth studies demonstrated that RV-EC were capable of significant growth when maintained in serum-free conditions. Growth kinetics using serum-free conditioned medium demonstrated mitogenic activity indicating the presence of an autocrine growth factor. Increase growth responsiveness was also noted in RV-EC when treated with a variety of peptide growth factors. These results indicate that resistance vessel endothelium can be successfully isolated and maintained in long-term serial cultures. Furthermore, the availability of cultured EC and SMC from this unique microvascular site will enable examination of cerebrovascular endothelial-smooth muscle cell interactions in vitro and may help to elucidate the mechanisms of altered vascular function in disease states.

SUBPOPULATIONS OF BLOOD LYMPHOCYTES DEMONSTRATED BY QUANTITATIVE CYTOCHEMISTRY

The cell coats of glutaraldehyde-fixed blood lymphocytes from guinea pigs have been stained with Thorotrast and examined by electron microscopy. The lymphocytes have been classified into three groups with respect to the concentration and distribution of colloidal particles over their surfaces. The average number of particles per micron length of cell surface membrane, the particle density, has then been measured for each class of cells. These values are distributed into two clusters separated by a gap. A new statistical test for bimodality has been devised to evaluate the significance of such a gap in terms of its dip intensity. The results of this analysis demonstrate the existence of at least two distinct populations of blood lymphocytes, indistinguishable by other morphologic criteria.

Ultrastructure of coronary arteries and myocardium in experimental hypertension.

Abstract The ultrastructure of coronary arteries and myocardium has been examined after 1–4 weeks of experimental hypertension produced in Wistar-Kyoto rats by unilateral renal artery constriction. Upon sacrifice blocks of ventricular myocardium were processed for light and electron microscopy. Two types of medial necrosis are observed in epicardial arteries: one involving elongated smooth muscle cells throughout the media and the other localized to the outer layers of the vessel wall. Some of the epicardial and intramyocardial arteries exhibit hyperplastic lesions characterized by smooth muscle cells in the subendothelial space. Medial smooth muscle cells of small intramyocardial arteries also exhibit contraction, fragmentation and necrosis. Plasma, cells and fibrin accumulate in the subendothelial space of intramyocardial arteries. Discrete areas of myocardial injury of variable severity are present in the subendocardium of the left ventricle. The vascular lesions in epicardial and intramyocardial arteries are related to the severity of blood pressure elevation and the vasoconstrictor actions of angiotensin II, while the foci of myocardial damage are consistent with ischemic or anoxic injury.

Cerebrovascular angiotensin II receptors in spontaneously hypertensive rats

The objective of this study was to characterize angiotensin II (AII) receptors in cerebral capillary endothelium and to examine whether the first step in All responsiveness, namely All receptor binding, is aberrant in cerebral microvessels obtained from adult spontaneously hypertensive rats (SHR). The binding of [3H]angiotensin II to isolated cerebrocortical microvessels from Sprague-Dawley, Wistar-Kyoto, and SHR rats was used to characterize All receptors on these vessels. Kinetic experiments yielded an equilibrium-derived Kd (dissociation rate constant/association rate constant) very close to that obtained from Scatchard analysis of saturation binding data. The data indicated that the two normotensive control strains exhibited comparable All receptor affinity and binding capacity. In contrast, experiments with microvessels from adult SHR indicated a significantly higher sbiBmax for All receptors relative to controls. Although experiments assessing functional endothelial alterations in the SHR to All remain to be performed, the increase in All receptor number suggests that an abnormality in vascular AII responsiveness may play an important role in this model of hypertension.

Pyruvate dehydrogenase activity in cardiac mitochondria from genetically diabetic mice

Pyruvate dehydrogenase (PDH) was studied in isolated heart mitochondria from genetically diabetic mice (C57BL/KsJ db/db) with progressive cardiomyopathy. Both the basal activity (active enzyme) and the total activity (the sum of active and inactive enzyme) were determined. In mitochondrjal extracts from 8–18-wk-old db/db mice, there was a 73% decrease of basal activity accompanied by a 38% decrease of total activity as compared with controls. The lower basal activity at 8 wk of age suggests an increased conversion of the enzyme into the phosphorylated(inactive) form. Evidence is also given that the conversion of inactive (phosphorylated) enzyme into active (dephosphorylated) enzyme is inhibited in cardiac mitochondria prepared from 8-wk and older db/db mice. These changes coincide with the onset of defective oxidative metabolism andcan explain the depressed pyruvate oxidation reported previously.

Selective expression of c-mas proto-oncogene in rat cerebral endothelial cells

NORTHERN blot analysis of cultured endothelial cells derived from rat cerebral resistance vessels demonstrated the presence of c-mas mRNA. This is the first description of mas expression in non-neuronal cells. c-mas message was not detectable in cultured endothelial cells derived from other vascular beds, including rat aorta and mesenteric resistance vessels. Since c-mas has been purported to regulate the proliferation response to angiotensin, the growth properties of all three endothelial cell cultures were examined. The data indicated no differences in either basal or angiotensin-stimulated cell growth among brain, mesenteric or aortic-derived endothelial cells. These data suggest that c-mas is selectively expressed in brain endothelial cells and that this proto-oncogene does not regulate cell proliferation in this cell type.

Regional variation in capillary permeability of ventricular myocardium.

Abstract The transcapillary exchange of horseradish peroxidase (HRP) has been investigated in the principal layers of rat ventricular myocardium. Cardiac arrest was induced in rats by the intravenous (iv) administration of KCl at intervals of 4–180 sec after the iv injection of HRP. The coronary arteries of non-working hearts were perfused with peroxidase for 90–300 sec. Sections from both ventricles were processed for light and electron cytochemistry, and thin sections examined by electron microscopy. The capillaries in the subendocardial and intermediate regions of both ventricles exhibit greater permeability than the subepicardial capillaries, as manifested by the more rapid extravascular passage of HRP. This permeability gradient is detectable for at least 15 sec after iv injection of the tracer, and for as long as 5 minutes when the enzyme is perfused. The observations suggest that permeability differences in subendocardial and subepicardial capillaries have an ultrastructural basis and provide further evidence for the functional heterogeneity of ventricular myocardium.

Cerebrovascular ultrastructure and permeability after carotid artery constriction in experimental hypertension

Abstract The purpose of the present study was to examine the effects of carotid artery constriction on cerebrovascular ultrastructure and permeability in severe two-kidney Goldblatt hypertension in the rat, a renin-angiotensin dependent state. The right common carotid artery of male Holtzman rats was constricted with a silver wire clip having an i.d. of 0.27 mm. Forty-eight hours later the left renal artery of these animals was similarly constricted. Severely hypertensive animals (b.p. > 200 mmHg) and controls were injected with horseradish peroxidase (HRP) and sacrificed 5–20 min later. Parietal cortex supplied by unconstricted and constricted carotid arteries was then processed for light and electron microscopy and cytochemistry. The cerebral cortical arterial vessels from the contralateral hemisphere of the hypertensive animals are tortuous and exhibit segmental constrictions. Ultrastructural examination reveals arterial hyalinosis and intramural and perivascular fibrin deposition. HRP penetrates interendothelial arterial junctions and is found within the subendothelial space, around medial smooth muscle cells and between swollen cell processes of the neuropile. The penetrating arterial vessels of the ipsilateral hemisphere also exhibit increased permeability to HRP but there are no morphologic alterations in the arterial vasculature or adjacent neuropile. Since carotid artery constriction effectively excludes elevated arterial pressure (intracarotid pressure ≤ 145 mmHg) as the cause of the abnormal cerebrovascular permeability, it is suggested that the increased permeability in this system is due to angiotensin-mediated contraction of cerebral arterial endothelium.

Interendothelial gap size of subendocardial vs subepicardial capillaries

Abstract The intercellular endothelial junctions of subendocardial and subepicardial capillaries of rat ventricular myocardium have been studied following in vitro exposure to lanthanum and uranyl acetate staining en bloc . While the intercellular clefts of subendocardial capillaries are uniformly filled by tracer particles, the interendothelial clefts of subepicardial capillaries are only partially permeated by the tracer. The width of the gaps at junctional areas in these capillaries has been determined in the uranyl acetate-treated material. Mean gap distances for subendocardial capillaries are significantly larger than those of subepicardial capillaries. The functional implications of these observations are discussed.