Modeling pulmonary fibrosis by abnormal expression of telomerase/apoptosis/collagen V in experimental usual interstitial pneumonia

Limitations on tissue proliferation capacity determined by telomerase/apoptosis balance have been implicated in pathogenesis of idiopathic pulmonary fibrosis. In addition, collagen V shows promise as an inductor of apoptosis. We evaluated the quantitative relationship between the telomerase/apoptosis index, collagen V synthesis, and epithelial/fibroblast replication in mice exposed to butylated hydroxytoluene (BHT) at high oxygen concentration. Two groups of mice were analyzed: 20 mice received BHT, and 10 control mice received corn oil. Telomerase expression, apoptosis, collagen I, III, and V fibers, and hydroxyproline were evaluated by immunohistochemistry, in situ detection of apoptosis, electron microscopy, immunofluorescence, and histomorphometry. Electron microscopy confirmed the presence of increased alveolar epithelial cells type 1 (AEC1) in apoptosis. Immunostaining showed increased nuclear expression of telomerase in AEC type 2 (AEC2) between normal and chronic scarring areas of usual interstitial pneumonia (UIP). Control lungs and normal areas from UIP lungs showed weak green birefringence of type I and III collagens in the alveolar wall and type V collagen in the basement membrane of alveolar capillaries. The increase in collagen V was greater than collagens I and III in scarring areas of UIP. A significant direct association was found between collagen V and AEC2 apoptosis. We concluded that telomerase, collagen V fiber density, and apoptosis evaluation in experimental UIP offers the potential to control reepithelization of alveolar septa and fibroblast proliferation. Strategies aimed at preventing high rates of collagen V synthesis, or local responses to high rates of cell apoptosis, may have a significant impact in pulmonary fibrosis.

Modeling pulmonary fibrosis by abnormal expression of telomerase/apoptosis/collagen V in experimental usual interstitial pneumonia.

Limitations on tissue proliferation capacity determined by telomerase/apoptosis balance have been implicated in pathogenesis of idiopathic pulmonary fibrosis. In addition, collagen V shows promise as an inductor of apoptosis. We evaluated the quantitative relationship between the telomerase/apoptosis index, collagen V synthesis, and epithelial/fibroblast replication in mice exposed to butylated hydroxytoluene (BHT) at high oxygen concentration. Two groups of mice were analyzed: 20 mice received BHT, and 10 control mice received corn oil. Telomerase expression, apoptosis, collagen I, III, and V fibers, and hydroxyproline were evaluated by immunohistochemistry, in situ detection of apoptosis, electron microscopy, immunofluorescence, and histomorphometry. Electron microscopy confirmed the presence of increased alveolar epithelial cells type 1 (AEC1) in apoptosis. Immunostaining showed increased nuclear expression of telomerase in AEC type 2 (AEC2) between normal and chronic scarring areas of usual interstitial pneumonia (UIP). Control lungs and normal areas from UIP lungs showed weak green birefringence of type I and III collagens in the alveolar wall and type V collagen in the basement membrane of alveolar capillaries. The increase in collagen V was greater than collagens I and III in scarring areas of UIP. A significant direct association was found between collagen V and AEC2 apoptosis. We concluded that telomerase, collagen V fiber density, and apoptosis evaluation in experimental UIP offers the potential to control reepithelization of alveolar septa and fibroblast proliferation. Strategies aimed at preventing high rates of collagen V synthesis, or local responses to high rates of cell apoptosis, may have a significant impact in pulmonary fibrosis.

Influence of a hypercholesterolemic diet on the collagen composition of the bladder wall extracellular matrix in rats.

PURPOSE: To investigate the effects of hypercholesterolemic diet on the collagen composition of urinary bladder wall. MATERIALS AND METHODS: Forty-five female 4-week-old Wistar rats were divided into three groups: 1) control group fed a normal diet (ND); 2) model of bladder outlet obstruction (BOO) group fed a ND; and 3) group fed a HCD (1.25% cholesterol). Total serum cholesterol, LDL cholesterol and body weight were assessed at baseline. Four weeks later, group 2 underwent a surgical procedure resulting in a partial BOO, while groups 1 and 3 underwent a sham similar surgical procedure. Six weeks later, all animals had their bladders removed; serum cholesterol and LDL cholesterol levels and body weights were measured. Morphological and morphometric analysis was performed by Picrosirius staining and collagen types I and III were identified by immunofluorescence. Statistical analysis was completed and significance was considered when p<0.05. RESULTS: Rats fed an HCD exhibited a significant increase in LDL cholesterol levels (p<0.001) and body weight (p=0.017), when compared to the groups fed a ND during the ten-week study period. Moreover, the HCD induced morphological alterations of the bladder wall collagen, regarding thin collagen fibers and the amounts of type III collagen when compared to the control group (p=0.002 and p=0.016, respectively), resembling the process promoted in the BOO model. CONCLUSIONS: A hyper-cholesterolemic diet in Wistar rats promoted morphological changes of the bladder types of collagen, as well as increases in body weight and LDL cholesterol.

Effect of topical clay application on the synthesis of collagen in skin: an experimental study.

BACKGROUND: Clay is often used in cosmetic treatments, although little is known about its action. AIM: To evaluate the effect of topical clay application on the histoarchitecture of collagen fibres in rat skin. METHODS: Animals received a daily application of clay and retinoic acid (RA) 0.025% to the dorsal skin over 7 and 14 days, under vaporization at 37 °C for 40 min. Control skin was not vaporized. Samples from each region were excised, and stained with picrosirius red for collagen evaluation. RESULTS: Seven days after clay treatment, an increase in the number of collagen fibres was observed in treated skin compared with control skin (51.74 ± 1.28 vs. 43.39 ± 1.79%, respectively, P < 0.01), whereas RA did not alter the collagen level (45.66 ± 1.10%). Clay application over 14 days did not induce a further increase in skin collagen, whereas treatment with RA did (58.07 ± 1.59%; P = 0.001 vs. control). CONCLUSION: Clay application promotes an increase in the number of collagen fibres, which may account for its beneficial effects.

The effects of conjugated estrogen, raloxifene and soy extract on collagen in rat bones.

OBJECTIVE: To evaluate the action of conjugated equine estrogen, raloxifene and isolated or combined genistein-rich soy extracts on collagen fibers in the bones of oophorectomized rats. MATERIALS AND METHODS: Seventy female rats received testosterone propionate (0.1 µg/g) on the 9th day after birth. At 6 months of age, the rats were administered the vehicle (propylene glycol, 0.5 ml/day), and ten of the rats were randomly chosen to comprise the non-oophorectomized control group (GI). The other 60 rats were ovariectomized and randomized into six groups of ten as follows: GII, vehicle; GIII, conjugated equine estrogen (CEE), 50 µg/kg/day; GIV, raloxifene (RAL), 0.75 mg/kg/day; GV, genistein-rich soy extract (GSE), 300 mg/kg/day; GVI, CEE + GSE, 50 µg/kg/day + 300 mg/kg/day; and GVII, CEE + RAL, 50 µg/kg/day + 0.75 mg/kg/day. Three months after surgery, the drugs were administered for 60 consecutive days. All rats were euthanized, and their left tibiae were removed for histological routine. The histological sections were stained with hematoxylin-eosin, and picrosirius for evaluating bone microarchitecture. Types I and II collagen fibers were analyzed by immunofluorescence. Data analysis was carried out with ANOVA and Tukey's test. RESULTS: Collagen reduction was significant in the GIII animals when compared to the other groups (p < 0.05). There was no significant difference in the thickness of collagen fibers among the groups. There was a greater quantity of type III collagen in GVI than in the other groups. CONCLUSION: Our data indicate that conjugated equine estrogen improves bone quality because it increases the quantity of type I collagen while reducing the quantity of thin collagen fibers. In addition, the combination of CEE and raloxifene or genistein-rich soy extract is not as efficient as CEE itself to improve bone quality.

Increased mRNA expression of collagen V gene in pulmonary fibrosis of systemic sclerosis.

BACKGROUND: Collagen V shows promise as an inducer of interstitial lung fibrosis in experimental systemic sclerosis (SSc). MATERIALS AND METHODS: Remodelling of the pulmonary interstitium was evaluated based on the clinical data and open lung biopsies from 15 patients with SSc. Normal lung tissues obtained from eight individuals who died of traumatic injuries were used as control group. Immunofluorescence, immunohistochemistry, morphometry, tri-dimensional reconstruction and a real-time polymerase chain reaction were used to evaluate the quantity, structure and molecular chains of collagen V. The impact of these markers was tested on clinical data. RESULTS: The main difference in collagen V content between SSc patients and the control group was an increased, abnormal and distorted fibre deposition in the alveolar septa and the pre-acinar artery wall. The lungs from SSc patients presented [alpha1(V)] and [alpha2(V)] mRNA chain expression increased, but [alpha2(V)] was proportionally increased compared with the control group. High levels of collagen V were inversely associated with vital capacity (r = -0.72; P = 0.002), forced vital capacity (r = -0.76; P < 0.001), forced expiratory volume in 1-s (r = -0.89; P < 0.001) and diffusing capacity for carbon monoxide (r = -0.62; P = 0.04). CONCLUSIONS: Abnormal collagen V fibres are overproduced in lungs from SSc patients and may play an important role in the pathogenesis of the disease as this molecule regulates tissue collagen assembly. The aberrant histoarchitecture observed in SSc can be related to the overexpression of the [alpha2(V)] gene of unknown origin.

Analysis of different staining techniques for c4d detection in renal allograft biopsies.

Capillary C4d deposition has been recognized as a marker of antibody-mediated rejection (AMR). Although the detection of capillary C4d by means of immunofluorescence (IF) in cryostat sections is well established, frozen tissue is not always available, thus limiting the diagnosis of AMR. The aim of the present study was to analyze different techniques for C4d staining and the prevalence of C4d in renal allograft biopsies. Detection of C4d was carried out using IF or immunohistochemistry (IHC) on frozen and paraffin sections of renal allograft biopsies available from the same patients. Biopsies obtained from 20 patients were classified into 3 groups: no rejection, acute rejection, and chronic allograft nephropathy (CAN). The capillary C4d deposition prevalence in frozen-IF, considered the gold standard technique for C4d detection, was 45% (9/20 cases). Compared with frozen-IF, the frozen-IHC technique presented an 85% concordance rate (17/20 cases; r = .70; P < .001; sensitivity = 77.8%; specificity = 90.9%). The paraffin-IF technique showed similar results, with an 80% concordance rate (16/20 cases; r = .64; P < .005; sensitivity = 55.6%; specificity = 100%), whereas C4d detection occurred in only 65% of paraffin-IHC cases (13/20; r = .30; not significant; sensitivity = 66.7%; specificity = 63.6%). No capillary C4d deposition was detected in cases without evidence of rejection. However, 4/7 cases (57%) of acute rejection were C4d positive. In the CAN group, 5/11 cases (45%) were C4d positive. In conclusion, these results demonstrated that frozen-IHC and paraffin-IF can be considered alternative techniques to frozen-IF for C4d detection. The paraffin-IHC technique displayed the lowest concordance rate for C4d detection.

Immune cell infiltration and broncovascular remodeling after nitric acid nasal instillation in a mouse bronchiolitis obliterans model.

Immune cell airway infiltration and the bronchovascular remodeling process have shown to be promising in the understanding of bronchiolitis obliterans (BO) pathogenesis. In this study we sought to validate the importance of immune cells, whether diffusely distributed or forming lymphoid follicles, collagen density, and vascular factors. Eight weeks after a single nitric acid (NA) nasal instillation, lung changes were characterized by lumen distortion, epithelial layer folding, reduction or total obliteration of terminal bronchiole (TB) lumen, and wall thickness increase. The morphologic changes in the TB and TA (terminal artery) lumen coincide with the measurement difference in the three groups. The TB diameter and lumen were significantly decreased in BO when compared with non-BO lungs (0.76 +/- 0.05 microm vs. 0.81 +/- 0.05 microm and 12,286.13 +/- 378.83 microm vs. 18,182.27 +/- 5,593.98 microm, p = 0.05 and p = 0.01, respectively). Equally significant was the increase in TB thickness in BO when compared with the non-BO group (201.72 +/- 35.75 microm vs. 149.75 +/- 40.61 microm, p = 0.007). The morphologic changes in immune cells seen in TB, TA, and bronchus-associated lymphoid tissue (BALT) also coincide with the quantification differences observed in the three groups. We concluded that immune cell infiltration and collagen/vascular remodeling are related to the spectrum of histologic changes in a BO nasal-induced model in mice and may be an appropriate target for prospective studies of human bronchiolitis.

Livaditis' circular myotomy does not decrease anastomotic leak rates and induces deleterious changes in anastomotic healing.

Considering that Livaditis' myotomy is still accepted as a good method for lengthening the esophagus to allow primary repair of long-gap esophageal atresia, the aim of this experimental study was to verify if this procedure decreases the incidence of leaks in anastomoses performed under severe tension. In addition, it was verified whether the myotomy promotes any morphological or biochemical change in the healing esophageal anastomosis. Sixty small dogs were submitted to a cervicotomy and resection of an esophageal segment (8.0 - 10.0 cm) resulting in an anastomosis under severe tension. The animals were divided into two groups (control group: only anastomosis; experimental group: anastomosis plus circular myotomy in the proximal esophageal segment). The animals were sacrificed on the 14th postoperative day, submitted to autopsy, and were evaluated as to the presence of leaks. Twelve scars of each group were collected for histological, histomorphometric (evaluation of scar thickness), electrophoretic and immunoblotting studies of collagen (total collagen and types of collagen determinations). Leak rates were the same in both groups. Histologic examination showed that the scar at the anastomosis was formed by fibrous tissue, without mucosa or muscular tissue. In the myotomy animals, a decreased number of newly formed small vessels was noted in comparison to control animals, and morphometric analysis showed that in the myotomy animals the anastomotic scar was thinner than in the control animals. Biochemical analysis of scars demonstrated that myotomy promoted a decrease in the soluble collagen content in comparison with the control animals and no alteration in the content of insoluble collagen. The electrophoretic separation of the types of collagen and characterization by immunoblotting demonstrated the presence of collagen types I, III, and V, and the quantification by densitometry of the bands showed a reduction in collagen type V (present in the blood vessels) in the myotomy animals in comparison to controls. This result is in accordance with the histological observation of a decrease in newly formed blood vessels. Circular myotomy does not decrease the possibility of anastomotic leaks, in addition to promoting deleterious changes in anastomotic healing.

Increase of interstitial collagen in the mouse endometrium during decidualization.

Decidualization in the mouse consists of an extensive remodeling of the endometrial extracellular matrix, resulting in a reduction of the extracellular spaces, an increase in the diameter of collagen fibrils, and changes in the relative ratio of different types of glycosaminoglycans. To assess the dynamic changes of the endometrial extracellular matrix during decidualization, collagen was analyzed biochemically and immunochemically in the endometrium of nulliparous and day 5 to day 8 pregnant mice. The amount of collagen per gram dry weight was higher in the endometrium of implantation sites than in interimplantation sites. Collagen types I, III, and V were the main components of the endometrium of nulliparous and pregnant animals. The amount of collagen type V was higher in the endometrium of pregnant animals than in nulliparous ones. A relative unusual homotrimeric form of collagen type V, probably formed by [alpha1(V)](3), was detected in pregnant endometrium by gel eletrophoresis and immunoblotting.

Changes in collagen matrix composition in human posterior tibial tendon dysfunction.

OBJECTIVE: To investigate whether tendon degeneration in posterior tibial tendon dysfunction syndrome is associated with changes in extracellular matrix collagen composition. METHODS: Specimens from grossly abnormal tendon regions from 9 patients with posterior tibial tendon dysfunction syndrome were prepared for routine histology. Collagens I, III and V were typed by immunoblotting and quantified by densitometry after SDS-PAGE. Proline and hydroxyproline residues were determined by liquid chromatography. Four other samples from grossly normal homologous tendon regions and one surgical specimen from a healthy patient undergoing arthrodesis of the ankle after an accident were included as control. RESULTS: In the grossly abnormal surgical posterior tibial tendon specimens we observed three types of histopathologic conditions present to varying degrees: increased mucin content, fibroblast hypercellularity and neovascularization. Analysis of degenerate tendons demonstrated a 79.3% increase in total proline and a 32.4% increase in 4-hydroxyproline. In addition, damaged tissue contained a higher proportion of collagen type III (mean increase: 53.6%) associated with a concomitant increase in type V collagen (mean increase: 26.4%). These alterations were accompanied by a reduction in type I collagen (mean decrease: 41.4%). CONCLUSIONS: In posterior tibial tendon dysfunction syndrome, the degenerative process results from marked changes in both structural organization and molecular composition of matrix collagens. The higher proportion of type V and type IlI collagens in degenerated tendons is likely to contribute to a decrease in the mechanical resistance of the tissue.

Very high collagen increase in placentae of smoking mothers.

In 55 placentae, collected at term from clinically normal pregnancies, hydroxyproline was assayed. Thirty were from mothers who smoke and 25 from nonsmokers. In placentae of smokers, hydroxyproline concentration was 42.77 +/- 8.77 micrograms/mg of tissue and in these of nonsmokers 22.52 +/- 7.46 micrograms/mg. Such high a difference discloses a large increment in placental collagen production in smokers, which may account for some of the noxious effects of tobacco on pregnancy.

PIP: Hydroxyproline was assayed in 55 placentae, collected at term from clinically normal pregnancies. 30 came from mothers who smoke and 25 from nonsmokers. In the placentae of smokers, hydroxyproline concentration was 42.77 +or- 8.77 mcg/mg of tissue and in those of nonsmokers, 22.52 +or- 7.46 mcg/mg. Such a high difference discloses a large increment in placental collagen production among smokers, which may account for some of the noxious effects of tobacco on pregnancy.

Short communication: placental collagen and premature rupture of fetal membranes.

Collagen content of 43 placentae from clinically normal females who went into labour spontaneously was determined by assaying hydroxyproline. In 23 cases the membranes ruptured before or at the beginning of labour, and in 20 cases the rupture was intrumentally provoked during the expulsive period. In the group with prematurely ruptured membranes, hydroxyproline concentration was 11.52 +/- 1.83 micrograms/mg lyophilized tissue, whereas in the other group concentration was 20.29 +/- 4.32 micrograms/mg tissue. These data disclose a much lower collagen placental content in the group with premature rupturing of the membranes.

Reduction of hydroxyproline content in the vessels of the human umbilical cord in premature rupture.

The hydroxyproline content of the amnion and the umbilical vessels obtained from cases in which the fetal membranes were instrumentally or prematurely ruptured was assayed. Hydroxyproline concentration (in micrograms/mg) in the latter group was 23.76, 16.79 and 17.86 in the amnion, artery and vein, whereas in the former the corresponding values were 42.20, 26.61 and 32.48. These results show that, in addition to a decrease in the amnion, hydroxyproline is lowered also in the umbilical vessels, suggesting that the reduction in the collagen in the fetal membranes is probably a particular manifestation of a general metabolic deficiency.