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Connective tissue remodeling in corneal and scleral wounds.

The fluorescent dye dichlorotriazinyl aminofluorescein will bind to amino groups of proteins covalently under physiological conditions. It has been used to dye the connective tissue around an ulcer or nonpenetrating, linear incision in the rabbit cornea and sclera, and the healing of the tissue has been examined up to 1 yr later. Sagittal sections were stained for light microscopy, and adjacent unstained sections were examined in the fluorescence microscope. The stained sections showed the reestablishment of the lamellar organization of the stromal collagen across the site of the incision; the fluorescence showed where the connective tissue that was present when the wound was made persisted, and thereby defined the limits of remodeling in the healing process. In Bowman's layer and the adjacent stroma, there was often an abrupt transition from fluorescent to new, undyed connective tissue. Deeper in the scar, and particularly around the ulcer, dark streaks were observed between the fluorescent lamellae, showing apparently new (non-fluorescent) tissue interdigitating with the old. These observations are discussed in relation to the mechanism of healing and the residual mechanical weakness that persists across scars in the cornea and sclera.

Animals↗

Effects of pioglitazone on adipose tissue remodeling within the setting of obesity and insulin resistance.

Obesity and dysfunctional energy partitioning can lead to the development of insulin resistance and type 2 diabetes. The antidiabetic thiazolidinediones shift the energy balance toward storage, leading to an increase in whole-body adiposity. These studies examine the effects of pioglitazone (Pio) on adipose tissue physiology, accumulation, and distribution in female Zucker (fa/fa) rats. Pio treatment (up to 28 days) decreased the insulin-resistant and hyperlipidemic states and increased food consumption and whole-body adiposity. Magnetic resonance imaging (MRI) analysis and weights of fat pads demonstrated that the increase in adiposity was not only limited to the major fat depots but also to fat deposition throughout the body. Adipocyte sizing profiles, fat pad histology, and DNA content show that Pio treatment increased the number of small adipocytes because of both the appearance of new adipocytes and the shrinkage and/or disappearance of existing mature adipocytes. The remodeling was time dependent, with new small adipocytes appearing in clusters throughout the fat pad, and accompanied by a three- to fourfold increase in citrate synthase and fatty acid synthase activity. The appearance of new fat cells and the increase in fat mass were depot specific, with a rank order of responsiveness of ovarian > retroperitoneal > subcutaneous. This differential depot effect resulted in a redistribution of the fat mass in the abdominal region such that there was an increase in the visceral:subcutaneous ratio, as confirmed by MRI analysis. Although the increased adiposity is paradoxical to an improvement in insulin sensitivity, the quantitative increase of adipose mass should be viewed in context of the qualitative changes in adipose tissue, including the remodeling of adipocytes to a smaller size with higher lipid storage potential. This shift in energy balance is likely to result in lower circulating free fatty acid levels, ultimately improving insulin sensitivity and the metabolic state.

Adipocytes↗

Estrogen opposes the apoptotic effects of bone morphogenetic protein 7 on tissue remodeling.

Interactions between estrogen and growth factor signaling pathways at the level of gene expression play important roles in the function of reproductive tissues. For example, estrogen regulates transforming growth factor beta (TGFbeta) in the uterus during the proliferative phase of the mammalian reproductive cycle. Bone morphogenetic protein 7 (BMP-7), a member of the TGFbeta superfamily, is also involved in the development and function of reproductive tissues. However, relatively few studies have addressed the expression of BMP-7 in reproductive tissues, and the role of BMP-7 remains unclear. As part of an ongoing effort to understand how estrogen represses gene expression and to study its interactions with other signaling pathways, chick BMP-7 (cBMP-7) was cloned. cBMP-7 mRNA levels are repressed threefold within 8 h following estrogen treatment in the chick oviduct, an extremely estrogen-responsive reproductive tissue. This regulation occurs at the transcriptional level. Estrogen has a protective role in many tissues, and withdrawal from estrogen often leads to tissue regression; however, the mechanisms mediating regression of the oviduct remain unknown. Terminal transferase-mediated end-labeling and DNA laddering assays demonstrated that regression of the oviduct during estrogen withdrawal involves apoptosis, which is a novel observation. cBMP-7 mRNA levels during estrogen withdrawal increase concurrently with the apoptotic index of the oviduct. Furthermore, addition of purified BMP-7 induces apoptosis in primary oviduct cells. This report demonstrates that the function of BMP-7 in the oviduct involves the induction of apoptosis and that estrogen plays an important role in opposing this function.

Amino Acid Sequence↗

Human and rat mesangial cell receptors for glucose-modified proteins: potential role in kidney tissue remodelling and diabetic nephropathy.

Advanced glycosylation endproducts (AGEs) are derived from the nonenzymatic addition of glucose to proteins. AGEs have been found to accumulate on tissue proteins in patients with diabetes, and their accumulation is thought to play a role in the development of diabetic complications. The finding that macrophages and endothelial cells contain AGE-specific receptors led us to examine whether mesangial cells (MCs) also possess a mechanism for recognizing and processing AGEs. Membrane extracts isolated from rat and human MCs were found to bind AGE-bovine serum albumin (BSA) in a saturable fashion, with a binding affinity of 2.0 +/- 0.4 x 10(6) M-1 (500 nM). The binding was specific for the AGE adduct, since AGE-modified collagen I and ribonuclease both competitively inhibited 125I-AGE-BSA binding to MC membranes, while the unmodified proteins did not compete. Binding of AGE proteins was followed by slow internalization and degradation of the ligand. Ligand blotting of MC membrane extracts demonstrated three distinct AGE-binding membrane proteins of 50, 40, and 30 kD. Growth of MCs on various AGE-modified matrix proteins resulted in alterations in MC function, as demonstrated by enhanced production of fibronectin and decreased proliferation. These results point to the potential role that the interaction of AGE-modified proteins with MCs may play in vivo in promoting diabetic kidney disease.

Adult↗

Actions of PPARgamma agonism on adipose tissue remodeling, insulin sensitivity, and lipemia in absence of glucocorticoids.

Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists improve insulin sensitivity and lipemia partly through enhancing adipose tissue proliferation and capacity for lipid retention. The agonists also reduce local adipose glucocorticoid production, which may in turn contribute to their metabolic actions. This study assessed the effects of a PPARgamma agonist in the absence of glucocorticoids (adrenalectomy, ADX). Intact, ADX, and intact pair-fed (PF) rats were treated with the PPARgamma agonist rosiglitazone (RSG) for 2 wk. RSG increased inguinal (subcutaneous) white (50%) and brown adipose tissue (6-fold) weight but not that of retroperitoneal (visceral) white adipose tissue. ADX but not PF reduced fat accretion in both inguinal and retroperitoneal adipose depots but did not affect brown adipose mass. RSG no longer increased inguinal weight in ADX and PF rats but increased brown adipose mass, albeit less so than in intact rats. RSG increased cell proliferation in white (3-fold) and brown adipose tissue (6-fold), as assessed microscopically and by total DNA, an effect that was attenuated but not abrogated by ADX. RSG reduced the expression of the glucocorticoid-activating enzyme 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD1) in all adipose depots. RSG improved insulin sensitivity (reduction in fasting insulin and homeostasis model assessment of insulin resistance, both -50%) and triacylglycerolemia (-75%) regardless of the glucocorticoid status, these effects being fully additive to those of ADX and PF. In conclusion, RSG partially retained its ability to induce white and brown adipose cell proliferation and brown adipose fat accretion and further improved insulin sensitivity and lipemia in ADX rats, such effects being therefore independent from the PPARgamma-mediated modulation of glucocorticoids.

11-beta-Hydroxysteroid Dehydrogenases↗

Integrin-extracellular matrix interactions in connective tissue remodeling and osteoblast differentiation.

The differentiaton of bone cells is a complex multistep process. Bone is somewhat unusual in that it is very actively and continually remodeled in the adult and that maintenance of its mass in the mature organism is exquisitely sensitive to mechanical as well as chemical signals. Bone is also unique because it consists of a very large amount of extracellular matrix (ECM) that is mineralized. The integrin family of ECM receptors has been shown to play an important role in tissue morphogenesis in several systems. Our studies on the regulation of matrix remodeling enzymes by integrins in rabbit synovial fibroblasts show that two b1 integrin fibronectin (FN) receptor complexes (alpha 5 beta 1 and alpha 4 beta 1) cooperate in detecting subtle changes in the composition of the ECM. As a result of signal transduction by these integrins, the levels of mRNA and protein for several members of the metalloproteinase family are regulated in these cells. We have also used antibody and RGD peptide perturbation studies to determine the significance of cell/ECM interactions to normal osteogenesis. We found that interactions between the cell binding domain of FN and integrins are required for both normal morphogenesis and gene expression in cultured osteoblasts that differentiate to form bone-like tissue in culture. These data lead us to propose that beta 1 integrins play an important role in osteoblast differentiation as well as in bone remodeling.

Alkaline Phosphatase↗

Advanced glycosylation endproduct-specific receptors on human and rat T-lymphocytes mediate synthesis of interferon gamma: role in tissue remodeling.

During normal aging and in chronic diabetes the excessive accumulation of reactive glucose-protein or glucose-lipid adducts known as advanced glycosylation endproducts (AGEs) has been shown to induce tissue dysfunction, in part through interaction with AGE-specific receptors on monocyte/macrophages and other cells. Recognizing that circulating lymphocytes trafficking through tissues interact with tissue AGEs, we searched for the expression of AGE-binding sites on peripheral blood T lymphocytes. Resting rat and human T cells bound 125I-AGE-albumin with an affinity of 7.8 x 10(7) M-1, whereas, after stimulation with phytohemagglutinin (PHA) for 48 h, binding affinity increased to 5.8 x 10(8) M-1. Flow cytometric analysis of resting rat T cells using polyclonal antibodies raised against rat liver AGE-binding proteins (p60 and p90) revealed the constitutive expression of both immunoreactivities. The number of resting CD4+ and CD8+ T cells positive for anti-p60 antibody binding (34.2 and 58.5%, respectively) increased to 92 and 90% of cells after 48-h stimulation with PHA. Exposure of PHA-activated T lymphocytes to AGE-albumin enhanced expression of interferon gamma (IFN-gamma) mRNA 10-fold and induced greater elaboration of the mature protein than did exposure to unmodified protein or PHA treatment alone. These data indicate that T cells contain an inducible system of surface receptors for AGE-modified proteins, and that receptor occupancy is linked to lymphokine production. This T cell AGE-receptor system might serve to target lymphocytes to AGE-rich tissues and involve them in the regulation of tissue homeostasis either by assisting in macrophage-dependent clearance of AGE-proteins, or by exerting direct antiproliferative action on mesenchymal cells. Under conditions of excessive AGE-protein and AGE lipid accumulation (e.g., aging and diabetes), enhanced production of AGE-induced IFN-gamma may accelerate immune responses that contribute to tissue injury.

Animals↗

Effect of steroid hormones on tissue remodelling and progesterone receptors in the uterus of seasonally anoestrous brushtail possums (Trichosurus vulpecula).

This study describes progesterone receptor (PR) location within uterine cells and associated morphological changes to the uterine luminal and glandular epithelium in seasonally anoestrous brushtail possums treated with oestradiol and/or progesterone. Twenty-four adult female possums (n = 6/group) were treated with oestradiol, progesterone, oestradiol followed by progesterone or with the oil vehicle alone for 6-day periods. Uterine tissue was recovered, weighed and processed for light and transmission electron microscopy and for immunohistochemistry for PRs. Stereological techniques were used to quantify epithelial cell and constituent volumes for both luminal and glandular tissues. Plasma concentrations of oestradiol and progesterone were determined by radioimmunoassay. Mean uterine wet weights were significantly heavier (P < 0.001) following oestradiol/progesterone treatment and maximum gland dilation, cellular and stromal growth, maximum cell height, and cell and constituent volumes were recorded after this treatment regimen. Cell nuclei and debris were commonly observed in gland lumina, and nuclear PRs were found predominantly in stromal cells following oestradiol-only treatment. Sequential treatment with oestradiol and then progesterone caused a decline in the number of positively stained cells. Epithelial cells contained extensive secretory organelles and degenerating cells were common within the glands. Oestradiol treatment induced cell and cell constituent growth and promoted PR formation in anoestrous possum uterine tissue. Subsequent exposure to progesterone stimulated uterine tissues to reach maximum wet weights and led to the cellular maturation necessary to remodel the uterus in preparation for pregnancy.

Anestrus↗

Matrix metalloproteinases-2 and -9 and their endogenous tissue inhibitors in tissue remodeling after sealing of the fetal membranes in a sheep model of fetoscopic surgery.

OBJECTIVES: We studied collagen plugging of the fetoscopic access site in an in vivo fetal lamb model for fetoscopic surgery and possible role for matrix metalloproteinase (MMP)-2 and -9 and tissue inhibitors (TIMPs). METHODS: Eight ewes had fetoscopic balloon occlusion of the trachea as an experimental treatment for congenital diaphragmatic hernia between days 88 and 99 of gestation (term 145 days) with sampling of amniotic, allantoic, and tracheal fluid. Nonoperated cotwins were used as controls. The fetoscopy port was closed using a collagen plug. Ten days (range 9-12) later, fluids were sampled and plug sites collected for histologic analysis. Activity of MMP-2 (72 kDa, gelatinase A) and MMP-9 (92 kDa, gelatinase B) was determined in the fluids by zymography and secretion of TIMPs (27-30 kDa; TIMP-1, glycosylated TIMP-3 and TIMP-4, 24 kDa; unglycosylated TIMP-3, 21 kDa; TIMP-2) by reverse zymography and quantified by densitometric analysis. RESULTS: No pregnancy was complicated by amniorhexis or preterm labor. At cesarean, normal volumes of amniotic and allantoic fluid were present in all cases. Histology of the plug sites revealed good integration of the collagen plug without complete restoration of membrane integrity. MMP-2, MMP-9, and TIMPs were detected in all fluids. In the operated animals, significantly (P <.05) higher activity of MMP-9 was found in amniotic fluid, with lower concentrations of TIMPs in allantoic fluid (P <.01). Tracheal occlusion was associated with a significant (P <.02) increase in both MMP-2 and -9 in tracheal fluid. CONCLUSION: Collagen plugging of the fetoscopic access port sites in sheep resulted in functionally effective sealing of the fetal membranes. Changes in MMP-2, MMP-9, and TIMPs suggest an active remodeling of both the fetal lung and the fetal membranes.

Amnion↗

Tissue remodeling in Guinea pig lateral prostate at different ages after estradiol treatment.

Estrogen seems to have an essential role in the fibromuscular growth characteristic of benign prostatic hyperplasia (BPH). This paper describes the effects of chronic estradiol treatment on Guinea pig prostatic stroma at different ages. Tissues from experimental animals were studied by histological and histochemical procedures, morphometric-stereological analysis and transmission electron microscopy (TEM). Marked fibromuscular hypertrophy was observed after estradiol treatment in animals of pre-pubertal and adult ages. Increases in the density and thickness of the collagen and elastic fibers were observed by histochemistry. TEM revealed wide distributions of collagen fibrils and large elastic fibers adjacent to the epithelial basal lamina and between the stromal cells, establishing contacts between them. These results indicate that the Guinea pig prostate simulates the stromal modifications observed in BPH in some aged animals after estrogen treatment at different ages, making it a good model for this disease.

Age Factors↗

Rapid new bone tissue remodeling during distraction osteogenesis is associated with apoptosis.

During the process of distraction osteogenesis new bone forms and undergoes rapid remodeling. Apoptosis may be one of the regulatory mechanisms governing the removal of the redundant callus during distraction osteogenesis. A rabbit tibial lengthening model was used and lengthened at 0.7 mm/day for 3 weeks. The regenerating tissues from the distraction gap were examined for apoptotic changes by transmission electron microscopy (TEM) and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method. Osteoclastic bone resorption activities were demonstrated by tartrate resistant acid phosphatase (TRAP) staining. The apoptotic cells were mainly present in the transitional regions between the fibrous tissue and the new bone in the mineralization front, and close to or on the new bone surfaces near the center of the regenerate. The TUNEL labeling was greatly reduced in the mature bone near the osteotomied bone ends. TEM examination confirmed the presence of cells with apoptotic changes at various regions of the regenerate. TRAP staining revealed that osteoclastic bone resorption activities in the regenerate were in a similar pattern of distribution to those of the TUNEL labeling. The localization of apoptotic cells at the different regions of the regenerate, accompanied by the osteoclast activities, suggest that apoptosis is closely related to bone formation and remodeling during distraction osteogenesis.

Animals↗

Distribution of particulate matter and tissue remodeling in the human lung.

We examined the relationship between intrapulmonary particle distribution of carbonaceous and mineral dusts and remodeling of the airways along anatomically distinct airway paths in the lungs of Hispanic males from the central valley of California. Lung autopsy specimens from the Fresno County Coroner's Office were prepared by intratracheal instillation of 2% glutaraldehyde at 30 cm H(2)O pressure. Two distinct airway paths into the apico-posterior and apico-anterior portions of the left upper lung lobe were followed. Tissue samples for histologic analysis were generally taken from the intrapulmonary second, fourth, sixth, and ninth airway generations. Parenchymal tissues beyond the 12th airway generation of each airway path were also analyzed. There was little evidence of visible particle accumulation in the larger conducting airways (generations 2-6), except in bronchial-associated lymphoid tissues and within peribronchial connective tissue. In contrast, terminal and respiratory bronchioles arising from each pathway revealed varying degrees of wall thickening and remodeling. Walls with marked thickening contained moderate to heavy amounts of carbonaceous and mineral dusts. Wall thickening was associated with increases in collagen and interstitial inflammatory cells, including dust-laden macrophages. These changes were significantly greater in first-generation respiratory bronchioles compared to second- and third-generation respiratory bronchioles. These findings suggest that accumulation of carbonaceous and mineral dust in the lungs is significantly affected by lung anatomy with the greatest retention in centers of lung acini. Furthermore, there is significant remodeling of this transitional zone in humans exposed to ambient particulate matter.

Adolescent↗

Collagenase 2 (MMP-8) expression in murine tissue-remodeling processes. Analysis of its potential role in postpartum involution of the uterus.

Neutrophil collagenase or collagenase 2 (MMP-8) is unique among the family of matrix metalloproteinases (MMPs) because of its exclusive pattern of expression in inflammatory conditions. At present, no evidence of the occurrence of this enzyme in tissues other than human has been reported. In this work, we have cloned the murine homologue of human collagenase 2. The isolated cDNA contains an open reading frame coding for a polypeptide of 465 amino acids, which is 74% identical to its human counterpart. The mouse collagenase 2 exhibits the domain structure characteristic of several MMPs, including a signal sequence, a prodomain with the cysteine residue essential for enzyme latency, an activation locus with the Zinc-binding site, and a COOH-terminal fragment with sequence similarity to hemopexin. It also contains the three conserved residues (Tyr-209, Asp-230, and Gly-232) located around the Zinc-binding site and are distinctive of the collagenase subfamily. Northern blot analysis of RNAs isolated from a variety of mouse tissues revealed that collagenase 2 is expressed at late stages during mouse embryogenesis, coinciding with the appearance of hematopoietic cells. In addition, collagenase 2 was highly expressed in the postpartum uterus starting at 1 day postpartum and extending up to 5 days. Enzymatic analysis revealed that matrilysin, another MMP overexpressed in uterine tissue, is able to activate murine procollagenase 2. These data suggest that both enzymes could form an activation cascade resulting in the generation of the collagenolytic activity required during the process of massive connective tissue resumption occurring in the involuting uterus.

Amino Acid Sequence↗

Lack of antigen-specific tissue remodeling in mice deficient in the macrophage galactose-type calcium-type lectin 1/CD301a.

Macrophage galactose-type C-type lectins (MGLs), which were recently named CD301, have 2 homologues in mice: MGL1 and MGL2. MGLs are expressed on macrophages and immature dendritic cells. The persistent presence of granulation tissue induced by a protein antigen was observed in wild-type mice but not in mice lacking an endogenous, macrophage-specific, galactose-type calcium-type lectin 1 (MGL1) in an air pouch model. The anti-MGL1 antibody suppressed the granulation tissue formation in wild-type mice. A large number of cells, present only in the pouch of MGL1-deficient mice, were not myeloid or lymphoid lineage cells and the number significantly declined after administration of interleukin 1 alpha (IL-1alpha) into the pouch of MGL1-deficient mice. Furthermore, granulation tissue was restored by this treatment and the cells obtained from the pouch of MGL1-deficient mice were incorporated into the granulation tissue when injected with IL-1alpha. Taken together, MGL1 expressed on a specific subpopulation of macrophages that secrete IL-1alpha was proposed to regulate specific cellular interactions crucial to granulation tissue formation.

Air↗

Involvement of matrix metalloproteinases 2 and 9, tissue inhibitor of metalloproteinases and apoptosis in tissue remodelling in the sheep placenta.

Placental growth and development is crucial for successful pregnancy. The aim of this study was to characterize the activity and localization of the matrix metalloproteinase 2 (MMP-2) and MMP-9, which are capable of degrading basement membrane collagen (predominantly collagen type IV), and their endogenous tissue inhibitor of matrix metalloproteinases (TIMPs), in amniotic fluid and in the developing ovine placenta. Cell deletion by apoptosis during placental development was also examined. Zymography with gelatin as substrate indicated that MMP-2 (72 kDa gelatinase A; predominantly latent form) was present in increasing amounts in amniotic fluid from day 70 of gestation to labour (days 140-145), and MMP-9 (92 kDa gelatinase B; predominantly latent form) was detectable from day 125 to labour; there was no increase in MMP-2 or -9 in labour. A broad range of TIMPs was detected in amniotic fluid; the molecular masses corresponded to TIMP-1, -2 and -3. Immunohistochemical techniques localized MMP-2, MMP-9 and TIMP-3 in the sheep placenta, predominantly in the trophoblast layer in uninucleate, but not binucleate, cells. However, MMP-2 and -9 activated proteins in placental homogenates were low throughout pregnancy. Apoptosis was identified by morphological criteria and also by TdT-mediated dUTP nick end labelling. Apoptosis was present in discrete regions in the placenta, predominantly in trophoblast cells near the tips and the basal regions of the fetomaternal interdigitations. During pregnancy the sheep placenta becomes more complex and the area of the fetomaternal interface increases. MMP-2 and -9 are likely to be involved in breaking down basement membranes to allow cell migration during this process. It is suggested that digestion of supporting extracellular matrix may trigger apoptosis and in some way increase the branching pattern in the villi.

Amniotic Fluid↗