Morphology and metabolism of an aortic intima-media preparation in which an intact endothelium is preserved.

Abstract

A B S T R A C T An in vitro preparation of rabbit aortic "intima-media" previously shown to exhibit stable rates of respiration and glucose metabolism and the high rate of aerobic glycolysis considered characteristic of the metabolism of this tissue was subjected to electron microscopic examination. In samples examined immediately after the aortae were dissected free of adipose tissue and adventitia, under conditions similar to those now in common use, marked and widespread alterations in endothelial cell structure were present, including loss of cell integrity. The vascular smooth muscle cells retained a normal electron microscopic (EM) appearance. During subsequent incubation with 5 mM glucose in KrebsRinger bicarbonate (KRB), pH 7.4, under the conditions usually employed in studies of this preparation, large zones of the luminal surface were rapidly denuded of endothelium, and the remaining endothelial cells exhibited a wide range of ultrastructural alterations. The smooth muscle cells, however, continued to maintain a normal EM appearance.A method was developed to prepare segments of rabbit aortic intima-media which retained an intact layer of endothelium resembling that observed in tissue fixed in situ. During a 1-h incubation with 5 mM glucose in KRB, pH 7.4, gas phase 5% C02/95% 02, containing 6% bovine serum albumin, the intact aortic intima-media preparation retains an essentially unmodified EM appearance and exhibits linear rates of respiration. Under these conditions the intact aortic intima-media preparation exhibits significantly higher rates of 02 uptake and glucose uptake than those observed in our previous prep- aration or in other reported aortic intima-media preparations. The intact aortic intima-media does not exhibit the high rate of aerobic glycolysis during in vitro incubation that has been considered characteristic of the metabolism of rabbit, rat, and swine aortic intima-media. In addition, the magnitude of the Pasteur effect was far greater than that observed in other aortic intima-media preparations. The data suggest that component cells of the aortic intima-media may derive a major fraction of their energy requirements from respiration; they raise further questions concerning the significance of the high rate of aerobic glycolysis observed when aortic intima-media preparations are incubated in vitro, and they suggest that documentation of the EM appearance of the endothelium in such preparations is desirable.

A system for long-term perfusion of rabbit aorta in vitro.

“…The oxygen consumption during the second day was calculated to be 23.9 nmol Cymg dry weight per hour. Morrison et al 41 determined the oxygen consumption in their intact intima + media preparation to be 39.5 nmol Oj/mg dry weight per hour (assuming dry weight/wet weight = 0.22), which is in good agreement with our value, considering that the viability was low in the outer one-third of our preparation. One way to get an approximation of the oxygen consumption in our system on a cellular basis might be to use DNA contents determined at similar perfusions (Figure 2A).…”

“…In short-term perfusions, most authors have relied upon a normal appearance of the endothelium in light microscopic sections or Hautchen preparations. 313234 However, it has been pointed out 41 that neither a satisfactory light microscopic appearance, nor a constant glucose consumption guarantees a preserved endothelium, as judged from ultrastructural evaluation. Consequently, it is obvious that a combined metabolic and morphological approach should be used in the assessment of tissue viability and, indeed, that endothelial integrity must be assessed by ultrastructural evaluation.…”

In vivo oxygen transport in the normal rabbit femoral arterial wall.

“…To make an approximate comparison, we have assumed tissue weight of 1.06 g/ml, with a water content of 75% (26,27). Using these values when necessary, representative results are 20 nlo/cm3-s wet tissue in human umbilical arteries (28); 50 nlo/cmn-s in bovine mesenteric veins (29); 57 and 71 nlO/cm3-s in rabbit aortas (5,11); and 110-230 nlO/cm113-s in resting and stimllulated dog femoral artery segments (30). Our meani value of 99 and range of 56-142 nlo/CM3-s appear reasonable with respect to these respirometer studies.…”

“…The solution of the dif ferential equation for the oxygen concentration c at radius r between r0 and ri for these conditions and uniform w is: (c0 -c) ln (ri/r0) w = (4NiD(c0 -ci))/r". (5) In that the equation does not include convective transport within the tissue of the wall, we assumed that a good fit would tend to exclude this as an important mechanism for oxygen transport within the wall. This assumption is supported in a previous paper by Back (33).…”

“…Recent evidence suggests that aerobic metabolism meets a larger percentage of the energy requirement of the wall than earlier references indicated (5). Hypoxia produces arterial injury and accelerates atherosclerosis in rabbits.…”