Localization of collagen in the rat lung: biochemical quantitation of types I and III collagen in small airways, vessels, and parenchyma

The marked insolubility of pulmonary collagen has limited its accurate biochemical quantitation in small samples of lung and other tissues. We have recently developed a microassay based on radioisotope dilution techniques that we have used for the accurate determination of types I and III collagen in extremely small tissue samples. By applying this method to carefully dissected small airways and vessels and samples of parenchymal tissue of rat lungs, we have localized and quantitated biochemically the type I and III structural collagens of the lung. Large pulmonary arteries are the units richest in these interstitial collagen types on the basis of dried tissue weight (50 micrograms/100 micrograms dried tissue). Amounts of both types I and III collagen are considerably lower in the alveolar domain than in vessels and airways of the rat lung. The proportion of tissue composed of these collagen types decreases centripetally in rat pulmonary arteries, but increases in the bronchial tree. The relative proportions of type I and type III remain constant in all the structures tested. The higher total amount of collagen in the nonalveolar domain has implications for biochemical studies based on whole lung samples.     

Localization of collagen in the rat lung: biochemical quantitation of types I and III collagen in small airways,vessels, and parenchyma

The marked insolubility of pulmonary collagen has limited its accurate biochemical quantitation in small samples of lung and other tissues. We have recently developed a microassay based on radioisotope dilution techniques that we have used for the accurate determination of types I and III collagen in extremely small tissue samples. By applying this method to carefully dissected small airways and vessels and samples of parenchymal tissue of rat lungs, we have localized and quantitated biochemically the type I and III structural collagens of the lung. Large pulmonary arteries are the units richest in these interstitial collagen types on the basis of dried tissue weight (50 μg/100 μg dried tissue). Amounts of both types I and III collagen are considerably lower in the alveolar domain than in vessels and airways of the rat lung. The proportion of tissue composed of these collagen types decreases centripetally in rat pulmonary arteries, but increases in the bronchial tree. The relative proportions of type I and type III remain constant in all the structures tested. The higher total amount of collagen in the nonalveolar domain has implications for biochemical studies based on whole lung samples.     

Regulation of fibrillar collagen types I and III and basement membrane type IV collagen gene expression in pressure overloaded rat myocardium

Left ventricular hypertrophy is based on cardiac myocyte growth. The hypertrophic process can be considered heterogeneous based on whether it also includes a remodeling and accumulation of fibrillar types I and III collagens that are responsible for impaired myocardial stiffness. In the heart, the messenger RNA (mRNA) for fibrillar collagen types I and III has been detected only in cardiac fibroblasts, whereas mRNA for basement membrane collagen type IV is present in both fibroblasts and myocytes. We studied the early and long-term expression of these collagenous proteins in rat myocardium after abdominal aortic banding with renal ischemia. Complementary DNA probes for rat pro-alpha 2 (I), mouse type III and mouse type IV collagens, and chicken beta-actin were used. Northern and dot blot analysis on total RNA extracted from left ventricular tissue indicated a sixfold increase in steady-state levels of mRNA for collagen type I on day 3 of abdominal aortic banding, which had declined to control levels by day 7 where it remained rather constant at 4 and 8 weeks. Type III collagen showed a similar pattern of gene expression after banding. mRNA levels for type IV collagen, on the other hand, were elevated on day 1 after banding, returning to control at day 7 and remaining constant. Actin mRNA levels also increased on day 1 of banding, followed by a rapid return to control levels. Monospecific antibody to types I and III collagens and immunofluorescent light microscopy on frozen sections of the myocardium revealed that at 1 week after banding, the distribution and density of these collagens were similar to those of control animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of collagen types I, III, IV and V in the walls of human arteries

Using immunofluorescence the localization of I, II, IV, V type collagen in different layers of the artery wall was established. The adventitia was shown to contain only I and III type collagen. All collagen types studied were identified in the media. The structural organization and quantity of different types of collagen were found to depend on the artery caliber. The distribution of I, III, IV, V type collagen in the aortal intima is described. The localization of athrombogenic collagen of type IV and V in the area of the endothelial basal membrane is highlighted. The authors revealed the presence of I and III type collagen in the subendothelium and the age-related increase in interstitial collagen levels in the intima which is important for predicting thrombosis in deendothelization . Employing immunoelectron microscopy, previously undescribed forms of I, III, IV, V type collagen organization into microgranular structures were ascertained.     

Localization of cells containing LHRH-like mRNA in rat forebrain using in situ hybridization

Using in situ hybridization, we localized cells in the rat forebrain which contain mRNA that hybridizes with a radiolabeled, synthetic oligodeoxyribonucleotide (59-mer) complementary to human LHRH mRNA in the region which includes the coding sequence for the decapeptide. These brain areas have been shown previously to contain immunoreactive LHRH cell bodies.     

Localization of insulin receptor mRNA in rat brain by in situ hybridization

Insulin receptor mRNA was demonstrated in rat brain slices by in situ hybridization with three 35S-oligonucleotide probes and contact film autoradiography. Specificity was confirmed by showing that (a) excess unlabeled probe abolished the signal, (b) an oligonucleotide probe for rat neuropeptide Y mRNA showed a different distribution of hybridization signal, and (c) the distribution of insulin receptor binding was consistent with the distribution of insulin receptor mRNA. Insulin receptor mRNA was most abundant in the granule cell layers of the olfactory bulb, cerebellum and dentate gyrus, in the pyramidal cell body layers of the pyriform cortex and hippocampus, in the choroid plexus and in the arcuate nucleus of the hypothalamus.     

Localization of cells producing erythropoietin in murine liver by in situ hybridization

In situ hybridization using antisense RNA probes was used to localize cells that produce erythropoietin (EPO) in the livers of anemic transgenic mice expressing the human EPO gene and in livers of anemic nontransgenic mice. In transgenic mice bled from a hematocrit of 55% to one of 10%, hepatocytes surrounding central veins synthesized large amounts of human EPO mRNA. EPO-producing cells were very rare in the area of portal triads. In transgenic mice bled to a hematocrit of 20%, a similar number and distribution of cells contained human EPO mRNA as was found with a 10% hematocrit, but the cells were less heavily labeled, indicating increased EPO production per cell at 10% hematocrit as compared with 20% hematocrit. No human EPO mRNA was detected in the kidneys of anemic transgenic mice, although endogenous murine EPO mRNA was strongly expressed in cortical interstitial cells. In sections of livers from nontransgenic mice bled from a hematocrit of 45% to one of 10%, only isolated cells produced EPO. When the types of cells could clearly be identified, approximately 80% of these cells were hepatocytes, while 20% had a nonepithelial morphology and were located in or adjacent to the sinusoidal spaces. When the sense strand was used as the RNA probe for in situ hybridization, no labeled cells were seen in normal or anemic livers. These results demonstrate that hepatocytes are responsible for production of EPO in both transgenic and nontransgenic mice and that a second cell type that is similar in morphology to EPO-producing interstitial cells in the kidney also produces EPO in the livers of nontransgenic mice.     

Localization of erythropoietin synthesizing cells in murine kidneys by in situ hybridization

In situ hybridization was used to localize the cells that produce erythropoietin (EP) in anemic murine kidneys. Kidneys from anemic and nonanemic mice were fixed and processed for paraffin embedding. Sections were hybridized with a 35S-labeled RNA probe complementary to mRNA coding for EP. An uncommon, but specific type of cell was intensely labeled in the cortices of anemic kidneys. The labeled cells were clearly nonglomerular and nontubular. Their location outside of the tubular basement membrane was consistent with that of a subset of interstitial cells or capillary endothelial cells.     

Use of in situ hybridization to identify collagen and albumin mRNAs in isolated mouse hepatocytes.

We present a simple and improved method for in situ localization of albumin and collagen mRNAs in isolated mouse hepatocytes. The cells were isolated by collagenase perfusion, mincing, and differential centrifugation. Nick-translated 3H-labeled mouse albumin cDNA (pmalb-2) and chicken pro-alpha 2(I) collagen cDNA (pCg45) probes were then hybridized with the cells in silane-treated microcentrifuge tubes. The cells were transferred and fixed to a microscope slide and hybridization was evaluated semiquantitatively by counting exposure of grains in autoradiographic emulsion placed over the cells. With this method of in situ hybridization, all hepatocytes appear to have significant, but highly variable, amounts of albumin mRNA. In addition, type I procollagen mRNA appears to be present at low abundance in hepatocytes. These results indicate that in situ hybridization can effectively demonstrate the presence of specific low- or high-abundance mRNAs in isolated well-differentiated eukaryotic cells.     

Simultaneous synthesis of types I and III collagen by fibroblasts in culture.

Specific antibodies against types I and III collagens and procollagens were used to localize these proteins in cultured human cells. These studies indicate that the same cell makes both proteins. No type III procollagen synthesis was observed in cells from two patients with two patients with the Ehlers-Danlos type IV syndrome.     

The effects of NSAIDs on types I, II, and III collagen metabolism in a rat osteoarthritis model

The effects of long-term use of celecoxib, ibuprofen, and indomethacin on types I, II, and III collagen metabolism were evaluated in rat osteoarthritis (OA) model. One hundred and thirty wistar rats were randomly divided into 4 groups: the celecoxib group, the ibuprofen group, the indomethacin group, and the normal saline group. The osteoarthritis was induced by the excision of the left Achilles tendon. In the 3rd, 6th, and 9th?month of treatment after surgically induced osteoarthritis, the articular cartilage was observed with microscope using HE staining. The expression of proteoglycans was semiquantified using toluidine blue staining. And, the expressions of types I, II, and III collagen in chondrocytes were examined using immunohistochemistry. The results suggested that celecoxib had no remarkable effects on the expression of types I, II, and III collagen. Ibuprofen upgraded the expression of types I, II, and III collagen and increased the synthesis of collagen. Indomethacin suppressed the expression of type II collagen and enhanced the expression of types I and III collagen. Therefore, during the long-term use of NSAIDs in osteoarthritis, celecoxib may have no remarkable influences on collagen metabolism of the articular cartilage and may be the ideal choice in the treatment of chronic destructive joint disease when anti-inflammatory drugs need to be used for a prolonged period. Ibuprofen may be unfavorable, and indomethacin may be harmful to collagen metabolism in OA treatment.     

Autoantibodies to types I and IV collagen and heart valve disease in systemic lupus erythematosus/antiphospholipid syndrome

Clinical Rheumatology - Introduction/objectives autoantibodies to types I and IV collagen have been described in rheumatic fever and infective endocarditis. We tried to elucidate if an autoimmune...     

Localization of glucocorticoid receptor mRNA in the male rat brain by in situ hybridization.

The localization and distribution of mRNA encoding the glucocorticoid receptor (GR) was investigated in tissue sections of the adult male rat brain by in situ hybridization and RNA blot analysis. GR mRNA levels were measured by quantitative autoradiography with 35S- and 32P-labeled RNA probes, respectively. Strong labeling was observed within the pyramidal nerve cells of the CA1 and CA2 areas of the hippocampal formation, in the granular cells of the dentate gyrus, in the parvocellular nerve cells of the paraventricular hypothalamic nucleus, and in the cells of the arcuate nucleus, especially the parvocellular part. Moderate labeling of a large number of nerve cells was observed within layers II, III, and VI of the neocortex and in many thalamic nuclei, especially the anterior and ventral nuclear groups as well as several midline nuclei. Within the cerebellar cortex, strong labeling was observed all over the granular layer. In the lower brainstem, strong labeling was found within the entire locus coeruleus and within the mesencephalic raphe nuclei rich in noradrenaline and 5-hydroxytryptamine cell bodies, respectively. A close correlation was found between the distribution of GR mRNA and the distribution of previously described GR immunoreactivity. These studies open the possibility of obtaining additional information on in vivo regulation of GR synthesis and how the brain may alter its sensitivity to circulating glucocorticoids.     

Expression of fibrillar types I and III and basement membrane collagen type IV genes in myocardium of tight skin mouse

STUDY OBJECTIVE--The aim was to study the expression of fibrillar collagen types I and III and basement membrane type IV collagen in the heart of the tight skin mouse, a genetic mutant with collagen overproduction in various organs. DESIGN--Collagen gene expression was measured in the ventricular tissues of the heart of the tight skin mouse and the age matched homozygous (+/+) litter mates by the use of cDNA probes to alpha 2 (I), alpha 1 (III) and alpha 2 (IV) procollagen and northern and dot blot analysis. Collagen deposition was examined by immunofluorescent light microscopy using monospecific antibodies to types I, III and IV collagens. EXPERIMENTAL MATERIAL--Heterozygous male (TSK/+) and normal (+/+) mice, 1.5-2 months old of the C57BL/6 strain were used. The animals were anaesthetised and the hearts were rapidly excised and processed for RNA extraction and antibody staining. MEASUREMENTS AND RESULTS--The results of northern and dot blot analyses showed a 41% increase in mRNA level for collagen type I, a 63% increase in mRNA level for type III and a 33% increase in type IV collagen in the ventricular myocardium (right and left ventricles) of the tight skin mouse compared to its counterpart in age matched homozygous (+/+) litter mates. mRNA levels for beta actin showed no significant increase. Immunofluorescent light microscopy and monospecific antibodies to types I, III and IV collagens were used to examine collagen deposition. The results showed that collagen type I fibres are thicker and denser in perivascular areas of the tight skin mouse heart compared to normal heart. No abnormal accumulation of type III fibres was observed. CONCLUSIONS--The heart of the tight skin mouse may be an appropriate model for studying the up regulation of cardiac collagen gene expression and its potential contribution to myocardial diseases.     

Localization of renin gene expression to monkey ovarian theca cells by in situ hybridization.

To investigate the sites of renin gene expression and localization of renin in primate ovaries, five cynomolgus (Macaca fascicularis) and one rhesus (Macaca mulatta) monkey were treated with gonadotropins to induce multiple follicle development. One ovary was removed before hCG injection (1200 IU) from three monkeys and one ovary was removed 36 h after hCG administration from three monkeys. In three monkeys, the remaining ovary was removed 3, 5, and 7 days after injection of hCG. To detect and localize renin messenger RNA, 35S-radiolabelled 1.1 kb length complementary DNA and RNA probes of human renin were used for in situ hybridization. To compare the synthesis with the presence and the storage of renin or prorenin, renin antigen was assessed by immunohistochemistry in the same tissues using a polyclonal antibody against human renin (R15). Renin mRNA was detected by in situ hybridization only in ovaries collected within 5 days of exposure to hCG. All such ovaries exhibited a positive signal. Renin mRNA was localized to the theca interna and theca lutein cells. Positive cells were observed in a few growing antral follicles, in occasional mature preovulatory follicles, in corpus luteum, and most strikingly in atretic follicles. No signal was detected in primordial, primary, or in small antral follicles of ovaries exposed to hCG. In contrast with the in situ hybridization data, no signal was detected by immunohistochemistry using antirenin antibodies which exhibited a positive signal in monkey kidney. These results indicate that hCG turns on renin gene expression. Renin is synthesized without significant intracellular storage in monkey ovarian theca interna cells and in corpus luteum. The absence of storage of renin is consistent with the high concentrations of prorenin found in ovarian follicular fluid of hCG stimulated primates and with our knowledge of cellular renin processing which indicate that prorenin is secreted constitutively as it is synthesized.     

Demonstration by immunofluorescence that the same cells from chick embryo aortas synthesize elastin and collagen types I and III

Cells were isolated from the aortas of 17-day old chick embryos and they were stained with fluorescent antibodies specific for Type I collagen, Type I procollagen, Type III collagen, elastin and prolyl hydroxylase. The results indicated that the same cells simultaneously synthesize Type I procollagen, Type III procollagen and elastin. The synthesis of procollagens, and the presence of prolyl hydroxylase, in the same cells which synthesize elastin may well explain why elastin contains hydroxy-proline.