David J. Riley

Collagen and elastin metabolism in hypertensive pulmonary arteries of rats

We evaluated the processes controlling the accumulation of collagen and elastin in main pulmonary arteries of rats during an episode of hypoxic pulmonary hypertension. Explant cultures of main pulmonary arteries were incubated with [3H]proline to measure collagen and protein synthesis and percent collagen synthesis. Elastin synthesis was measured by [14C]valine incorporation into insoluble elastin. Relative collagen synthesis increased twofold (from 1.1 +/- 0.2 x 10(3) to 2.0 +/- 1.0 x 10(3) disintegrations per minute [14C]hydroxyproline/vessel/hr/mg protein), relative collagen synthesis doubled (from 2% to 4-5% of total protein synthesis), and elastin synthesis increased ninefold (from 0.4 +/- 0.2 x 10(4) to 3.6 +/- 0.6 x 10(4) dpm [14C]valine/vessel/hr/mg protein) in early hypertension. The level of pro alpha l(I) collagen RNA paralleled the relative collagen synthetic rate during the study period. Within 7 days of recovery from hypoxia, collagen and elastin contents were normal. We conclude that collagen and elastin in main pulmonary arteries are synthesized rapidly during an episode of hypoxic pulmonary hypertension and that collagen and elastin are rapidly removed from the hypertensive vessel during normoxic recovery.

Alternative Splicing of Rat Tropoelastin mRNA is Tissue-Specific and Developmentally Regulated

Sequence analysis of cDNA clones coding for rat tropoelastin previously has identified two variants that potentially corresponded to alternatively spliced tropoelastin mRNAs (Pierce et al., 1990). We have now used S1 nuclease protection analysis of total RNA from aorta, skin and lungs of 10-day and 6-week old rats to localize all sites of alternative splicing in the tropoelastin mRNA and to examine tissue-specific and developmental regulation of the use of these sites. This analysis revealed multiple sites of alternative splicing involving rat tropoelastin coding sequences corresponding to exons 12 through 15 of the bovine tropoelastin gene and a single site of alternative splicing at sequences corresponding to exon 33. Messenger RNAs from all three tissues at both developmental stages were alternatively spliced at the same sites; there was no evidence for the use of an alternative splice site unique to a particular tissue or developmental stage. However, both tissue-specific and developmentally regulated differences were apparent in the proportion of rat tropoelastin mRNA alternatively spliced at exon 33. Tropoelastin mRNA from the aorta and lungs of neonatal rats was alternatively spliced at exon 33 ten time more frequently than tropoelastin mRNA from skin. Between 10 days and 6 weeks of development, the use of this site of alternative splicing decreased by twenty-fold in RNA from skin, ten-fold in RNA from lungs and two-fold in RNA from aorta. In contrast, alternative splicing at exons 12 through 15 occurred in a small percentage of the mRNA and use of these sites exhibited minimal tissue-specific differences or developmental regulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Strain specific respiratory air space enlargement in aged rats

We studied lung structure and function in Fischer-344 and Sprague Dawley rats to compare the pathophysiologic features of the aged lung in animal strains. Both strains were maintained under identical conditions of minimal exposure to injurious environmental agents. We measured the number, size, and surface area of alveoli, pressure-volume characteristics and connective tissue content of lungs at midlife (12 or 14 months of age) and old age (24 months of age). Results showed differences in the older versus younger group of the Sprague Dawley strain as indicated by enlarged air spaces [154 +/- 21 (SEM) versus 118 +/- 13 micromicroliter] (p less than 0.05), increased collagen (hydroxyproline content 4.1 +/- 0.1 versus 3.0 +/- 0.1 mg/lung) (p less than 0.05), and a leftward shifted pressure-volume curve. There was no change in surface area or alveolar number. The structural lesions are consistent with air space enlargement with fibrosis and not emphysema. In contrast, no major changes were found in the lungs with age in Fischer-344 rats. We hypothesize that in the Sprague Dawley strain the aging process impairs the ability of the lung to maintain normal structure and function. Two strains of rats which differ pathologically in old age may be useful in the study of the effects of aging on the lung.

Liposome-entrapped antifibrotic agent prevents collagen accumulation in hypertensive pulmonary arteries of rats.

We studied the therapeutic efficacy of an intravenously injected antifibrotic agent encapsulated in liposomes on inhibiting collagen accumulation in hypertensive blood vessels. cis-4-Hydroxy-L-proline (cHyp) in liposomes was injected into rats exposed to 10% O2, and drug effect was evaluated by measuring right ventricular pressure and hydroxyproline content of the pulmonary artery. Right ventricular pressure was 11 +/- 1 mm Hg (mean +/- SEM) 5 days after a single intravenous injection of 200 mg/kg cHyp in liposomes compared with 14 +/- 1 mm Hg in rats injected with empty liposomes; hydroxyproline content was also reduced by cHyp treatment (87 +/- 6 versus 107 +/- 7 micrograms per vessel) (p less than 0.05 for both, n = 6-9). Injections of cHyp in liposomes every 5 days partially prevented hypertension and vascular collagen accumulation during a 3-week exposure to hypoxia, and the dose required was one tenth the dose of unencapsulated cHyp. Therapeutic doses of cHyp in liposomes injected for 6 months affected tensile properties of main pulmonary artery and aorta, but there were no apparent histological effects on other organs. Liposomes injected intravenously were identified in pulmonary artery endothelial cells. The prolonged effect of a single injection of cHyp in liposomes may be due to uptake of the liposomes by the endothelium. Liposome delivery of drugs to the arterial wall may be useful in the study and treatment of hypertensive vascular disease.

Treatment of experimental silicosis with antifibrotic agents

We tested the efficacy of 2 antifibrotic agents, the proline analogue cis-4-hydroxy-L-proline (cHyp) and the lathyrogen beta-aminopropionitrile (BAPN), on experimental silicosis in hamsters. Silica (75 mg) was instilled intratracheally, and 3 months later lung hydroxyproline content, the volume density of silicotic nodules in lung parenchyma, fluid-filled lung pressure-volume curves, body weight and survival were measured. Animals were injected with cHyp, 200 mg/kg body weight, or BAPN, 150 mg/kg body weight, twice daily for 3 months. Hydroxyproline contents (mg/lung) at 3 months were: control, 0.8 +/- 0.1; silica, 1.4 +/- 0.1 (P less than 0.05 compared to control); silica-cHyp, 1.2 +/- 0.2; silica-BAPN, 1.4 +/- 0.1 (both NS compared to silica). The volume density of granuloma (% of surface area) was: silica, 0.7 +/- 0.1; silica-cHyp, 5.9 +/- 1.0; silica-BAPN, 9.7 +/- 1.5 (both P less than 0.5 compared to silica). There was no difference among the groups as assessed by lung pressure-volume curves. No toxic effects were produced on the skeletal system as assessed by bone hydroxyproline content and skeletal roentgenograms. Final body weights (g) were: silica, 114 +/- 5; silica-BAPN, 108 +/- 6; silica-cHyp, 88 +/- 7 (the latter P less than 0.05 compared to silica). Survival (%) was: silica, 62%; silica-BAPN, 34%, silica-cHyp, 28% (both P less than 0.05 compared to silica). These data show that cHyp and BAPN treatment did not prevent silica-induced pulmonary fibrosis, led to more extensive silicotic nodules, and were toxic. Both cHyp and BAPN have some efficacy in other models of fibrosis, and the observations in the present study could be specific to silicosis in the hamster.

Remodeling of Rat Neonatal Pulmonary Artery: Role of Matrix Metalloproteinases

We studied whether rapid thinning of large pulmonary arteries of neonatal rats is associated with breakdown of collagen. Pulmonary artery extracts from fetal to 21 days of age were assayed for collagen content and matrix metalloproteinases. Within 3 days postpartum, no changes in collagen content, collagenolytic activity, or levels of stromelysin-l or gelatinase A were observed. After day 3, collagen content and total proteolytic activity increased with little change in matrix metalloproteinase expression. Thus, collagen was not degraded, and the late increases in collagen and total proteolytic activity were probably growth related. Unlike adult rats in which collagen is broken down after reversal of hypoxic pulmonary artery remodeling, collagen is not broken down in neonatal pulmonary arteries during adaptation to extrauterine life.

Flow cytometric determination of cell proliferation in hypertensive blood vessels

BACKGROUNDnMeasurement of vascular cell proliferation in animal models of hypertension is currently accomplished by demonstrating [(3)H]-thymidine ([(3)H]-dT) incorporation into DNA using autoradiography. This method, however, is labor intensive, requires radioactivity, and is limited by the inherent difficulty in discriminating labeled and unlabeled cells. To address these limitations, a flow cytometric-based method is described utilizing incorporation of 5-bromo-2-deoxyuridine (BrdU) into DNA of nuclei isolated from blood vessels.nnnMETHODSnPulmonary hypertension was induced in rats by exposure to 10% O(2) (hypoxia) for varying periods of time. Pulmonary arteries and aorta from rats injected with BrdU prior to sacrifice were isolated, fixed with 10% formalin, and digested with Protease XIV. The intact nuclei liberated by this treatment were successively treated with HCl/Triton X-100 and sodium borate. Processed nuclei were probed with a BrdU-specific fluorescein-conjugated antibody, and the percentage of BrdU staining cells was determined using flow cytometry.nnnRESULTSnAn approximately 20-fold increase in BrdU-positive cells at 3 days of hypoxia in pulmonary arteries (relative to control) with no change in aorta was observed. These results were similar to previous studies using [(3)H]-dT labeling.nnnCONCLUSIONSnFlow cytometric determination of cell proliferation in blood vessels is a simple, objective technique that may facilitate measurement of cell proliferation in animal models of vascular disease.