Timo Vihersaari

Energy metabolism of experimental wounds at various oxygen environments.

Energy metabolism of healing tissue was studied in experimental wounds of rats chronically breathing 11% O2, air or 55% O2. Increasing oxygen supply elevated both PO2 and PCO2 in the wound tissue. At the early phases of healing hypoxic wounds contained less DNA than normoxic or hyperoxic tissues. In hypoxia the accumulation of wound collagen was clearly retarded. Furthermore, tissue taken from wounds healing in hypoxic environments and tested ex vivo in air showed decreased capacity for glucose utilization, lactate production and oxygen consumption. Concentrations of AMP, ADP and ATP in repair tissue increased as healing progressed. The more oxygen available the higher the amounts of ADP and ATP. The AMP content was not affected by changes in local oxygen tension. These results support the earlier concept that the supply of oxygen in healing tissue may be rate-limitimg. Reduction of available oxygen either by systemic hypoxia or by increased diffusion distance impedes healing.

Radical mastectomy wound as a model for studies of human wound metabolism.

Summary The nutritive aspects of human wounds were investigated in twelve patients who had undergone radical mastectomy for mammary cancer. Three days after surgery, the drains were removed and wound fluid was allowed to accumulate under the flaps. At certain intervals respiratory gas tensions, hydrogen ion concentration, buffering capacity, and lactate/pyruvate ratio were determined by aspiration of fluid filling the dead space of the wound. Between the fifth and fourteenth days after operation, the mean PO 2 increased from 14 mm Hg to 22 mm Hg whereas the mean PCO 2 , pH, and buffer base remained essentially unchanged. The lactate/pyruvate ratio decreased by 40 per cent during this period. Changes in the measured parameters became more prominent when plotted against the volume of the aspirated fluid (= dead space). Increases in fluid volume from 15 to 100 ml to over 300 ml was associated with a decrease in the mean PO 2 from 28 to 2 mm Hg and a 100 per cent increase in the lactate/pyruvate ratio. Changes in pH and PCO 2 values were inversely proportional with PCO 2 the greatest in large dead space wounds. Skin slough occurred once. In this wound, the PO 2 varied between 2 and 4 mm Hg, and the lactate/pyruvate ratio was extremely high. It is concluded that adequate oxygen supply and drainage are fundamental, and, to a great extent, rate-limiting for the healing of dead space wounds.

Collagen synthesis in cells cultured from v. Recklinghausen's neurofibromatosis

SummarySubcutaneous tumors of a patient with v. Recklinghausens neurofibromatosis contained about 31% collagen calculated on the basis of lipid-free dry weight. Slices of the tumors synthesized collagen at a rate (4.7–8.5% from total protein) which was higher than that of the skin slices (2.8–5.9%). Neurofibromatosis cells were cultured from tumors of two patients. They synthesized relatively much more collagen than cultures of skin fibroblasts of the same patient or of healthy age-matched control persons.The second patients cultures were studied in detail. The cell densities of these cultures were higher and expressed more variation than the densities of control skin fibroblasts. Ion exchange cellulose chromatograms, SDS-polyacrylamide gel electrophoresis and 3-hydroxyproline analysis of the radioactive proteins made by the cultures indicate that most of the collagenous proteins resembled type I collagen. High proliferative capacity and high collagen synthesis of selected neurofibromatosis cells explains the growth of solid tumors.

A rapid assay to measure collagen synthesis in cell cultures

Limited pepsin digestion and precipitation of resistant parts of proteins with perchloric acid on glass-fiber filters has been used as a rapid way to determine the radioactive collagen secreted into fibroblast culture media. The specificity of the pepsin cleavage was tested by digesting [14C]- or [3H]proline- and [3H]tyrosine-labeled procollagens. Radioactivities obtained with this method were comparable with those obtained with collagenase digestions or hydroxyproline determinations. Dialysis of the samples is avoided and the radioactive collagen can thus be determined from the small medium samples obtained from microtest plates. The method was used to localize a collagen synthesis-increasing factor in preparative isoelectric focusing of microphage culture media.

Effect of changes in inspired oxygen tension on wound metabolism.

This work was prompted by earlier findings of the beneficial effect of increased oxygen supply on wound healing. Enzyme activities in the limiting step of glycolysis, citric acid cycle and pentose phosphate cycle were determined in cellulose sponge implants of rats chronically, breathing 12% O(2), air or 55% O(2.) Respiratory gas tensions and concentrations of pyruvate and lactate were measured in wound fluid aspirated from the implants. Significant portions of repair tissue exist in conditions of extremely low oxygen tension. Probably because all added oxygen is readily consumed, the wound fluid PO(2) increased only slightly in hyperoxic environment. The wound PCO(2) increased in parallel with the inspired PO(2), probably due to enhanced production of carbon dioxide. Hyperoxia shifted the wound metabolism from anaerobic towards aerobic glycolysis. This occurred concurrently with activation of citric acid cycle. Succinic dehydrogenase, a linking enzyme between citric acid cycle and electron transfer chain, also increased with increasing oxygen tension. This oxygen-induced metabolical change has been previously observed in many other tissues.

Collagen in human aorta. Changes in the type III/I ratio and concentration of the reducible crosslink, dehydrohydroxylysinonorleucine in ascending aorta from healthy subjects of different age and patients with annulo-aortic ectasia

The type III/I + III collagen ratio was studied in intima-medial samples of ascending aortas obtained from patients with the Marfan syndrome or other annulo-aortic ectasia (dilatation of the ascending aorta) and from control subjects, using electrophoretic analysis of cyanogen bromide peptides. The [3H]borohydride-reduced crosslinks of collagens were analysed by ion-exchange chromatography. Type III/I + III collagen ratios were twice as high in adult aortas as those found in skin samples of the same age. This ratio was lower in fetal and very young aortic samples and in 6-8 out of 12 pathological aortas (including one sample from a Marfan patient) when compared with adult controls. In contrast, the type III/I + III collagen ratio was high in fetal or very young skin and the values obtained from several patients did not differ from those of the control skin samples. In one pathological aorta out of six studied, the concentration of the reducible crosslink, dehydrohydroxylysinonorleucine, was higher than in controls, suggesting increased collagen synthesis or impaired maturation of collagen. These changes point to altered collagen metabolism in aortas of patients with annulo-aortic ectasia.

Desmosines in aneurysms of the ascending aorta (annulo-aortic ectasia)

Amino acid chromatography was used for determination of the elastin-specific amino acids desmosine and isodesmosine in acid hydrolyzates of intima-medial samples taken intraoperatively from aneurysms of human ascending aorta. Elastin concentration of the specimens was also estimated by hot alkali extraction followed by nitrogen determination of the extracted material and the insoluble residue. All patients studied had annulo-aortic ectasia i.e., dilatation of the aortic annulus and the ascending aorta. Two patients with the Marfan syndrome had low aortic elastin concentration determined by both methods. A third Marfan syndrome patient, youngest of the three, also had a slightly reduced concentration of elastin in the aorta. Aortic samples were studied from five patients who did not have the classical Marfan syndrome. Two patients of those five had decreased aortic elastin concentration. The change in elastin concentration was accompanied by high hydroxyproline/proline or hydroxylysine/lysine ratios which indicates that the proteins of the aneurysmatic aortic wall contained more collagen than the proteins of the control aortic wall. These findings point to a change in the structure or metabolism of elastin in the aortic wall in the Marfan syndrome and at least in some other patients with annulo-aortic ectasia.

Lysyl oxidase activity and synthesis of desmosines in cultured human aortic cells and skin fibroblasts: comparison of cell lines from control subjects and patients with the Marfan syndrome or other annulo-aortic ectasia

The activity of lysyl oxidase, the cross-linking enzyme of elastin and collagen, was measured in culture media of human skin fibroblasts, human aortic medial smooth muscle cells (SMCs) and adventitial fibroblasts using [3H]lysine-labelled elastin substrate. In addition, biosynthesis of isodesmosine and desmosine, the cross-linking amino acids of elastin, was studied by metabolic labelling with [14C]lysine and subsequent amino acid chromatography of protein hydrolysates. Lysyl oxidase activity in culture media of skin fibroblasts and aortic smooth muscle cells increased with the growth of the cell population and was at the highest level in cultures of high cell density. Lysyl oxidase activity in the aortic cell cultures was about three times that of skin fibroblasts. Aortic smooth muscle cells synthesized at least 100 times more desmosines than skin or adventitial fibroblasts. No differences were observed in lysyl oxidase activity and synthesis of desmosines between aortic smooth muscle cells or skin fibroblasts from patients with the Marfan syndrome or other annulo-aortic ectasia (dilatation of the ascending aorta) and the corresponding controls.

Arginine depletion in macrophage medium inhibits collagen synthesis by fibroblasts

Abstract In the present work we demonstrate that non-activated, cultured rat peritoneal macrophages deplete arginine from their culture medium and that the use of this medium in fibroblast cultures may lead to decreased synthesis of collagen by fibroblasts.