Regeneration of corneal tissue

Penetrating wounds in rabbit corneas heal to form an opaque tissue that eventually becomes transparent. DNA content, dry weight, water content, and collagen content of the tissue gradually become more like that of normal cornea. The healing tissues also synthesize low-sulfated keratan sulfate, hyaluronic acid, and heparan sulfate. These glycosaminoglycans are not found in normal adult corneas but have been reported in fetal corneas. Previous studies have shown that collagen from healing corneal wounds and collagen from fetal corneas have very similar cross-linking patterns, but these patterns are different from those in normal adult collagen. The similarities between collagen and glycosaminoglycans in healing corneal wounds and in fetuses suggest some recapitulation of ontogenetic processes. The biochemical sequence and eventual return of transparency to the rabbit cornea indicate a capability for true regeneration of stromal tissue in the rabbit.     

Mineral content of calcified tissues in cystic fibrosis mice

Although abnormal hard tissue mineralization is a recognized complication of cystic fibrosis (CF), the pathogenesis leading from the defective cystic fibrosis transmembrane conductance regulator (CFTR) protein is poorly understood. We hypothesized that CFTR plays a direct role in the mineralization of bone and teeth and tested the hypothesis using CF mouse models [CFTR(−) mice]. In vivo measurements by dual-emission X-ray absorpitometry (DEXA) indicated that bone mineral density (BMD) was reduced in CF mice as compared to gender-matched littermates. However, no change was evident after correction of BMD for the covariant of body weight. The latter finding was confirmed in isolated femurs and nasal bones by standard dry-ashing and instrumental neutron activation analysis (INAA). INAA of the continuously growing hypsodont incisor teeth from CFTR(−) mice revealed reduced Ca and normal P in the enamel layer—a finding consistent with changes in the deciduous teeth of CF children. Interestingly, enamel fluoride was increased in the CFTR(−) incisors and may associate with abnormal enamel crystallite formation. The iron content of the incisor enamel was reduced, explaining the loss of yellow pigmentation in CFTR(−) incisors. In contrast to the incisors, the mineral content of the slow-growing brachydont molar teeth was not different between CFTR(−) and CFTR(+) mice. It was concluded that CFTR does not play a direct role in the mineralization of bones or brachydont teeth in mice. Functional CFTR is apparently required for normal mineralization of the hypsodont incisors. However, multiple changes in the mineral composition of the CF incisors suggest an indirect role for CFTR, perhaps by maintaining a normal salivary environment for continuous tooth eruption. Preliminary reports published in Pediatric Pulmonology, 14, 253A (1997) and 15, 253A (1998).     

Regeneration of transgenic plants of Mexican lime from Agrobacterium rhizogenes-transformed tissues

Transgenic Mexican lime [Citrus aurantifolia (Christm.) Swing] plants were regenerated from tissues transformed by Agrobacterium rhizogenes strain A4, containing the wild-type plasmid pRiA4 and the binary vector pESC4 with nos-npt II and cab-gus genes. Transgenic shoots were generated by two different approaches. The first approach used internodal stem segments cocultured with A. rhizogenes. These were placed onto regeneration medium containing Murashige and Skoog salts and B5 organic compounds supplemented with 8 g ⋅ l^–1 agar, 7.5 mg ⋅ l^–1 6-benzylaminopurine, 1.0 mg ⋅ l^–1 -naphthaleneacetic acid, 300 mg ⋅ l^–1 cefotaxime and 80 mg ⋅ l^–1 kanamycin as a selective agent, and incubated under continuous light at 25 °C. Under these conditions, 76% of the explants produced shoots directly with no hairy root phase, with a mean of 1.3 shoots per explant, and 88% of these shoots were genetically transformed as determined by β-glucuronidase (GUS) assays. In the second approach, segments of transformed roots (15 mm long) obtained from internodal stem segments cocultured with A. rhizogenes were cultured on the above regeneration medium under similar conditions. Forty-one percent of these transformed root segments produced adventitious shoots, with a mean of 2.2 shoots per explant and with 90% of shoots transformed. GUS activity was evident in the transformed roots and in all parts of both transformed shoots and regenerated plants. The presence of the npt II and rolB genes in the regenerated plants was confirmed by PCR analysis. The presence of the npt II gene in the regenerated plants was also confirmed by Southern blot. Using these transformation systems, more than 300 Mexican lime transgenic plants were obtained, 60 of which were adapted to growing in soil. Received: 15 March 1997 / Revision received: 30 December 1997 / Accepted: 19 January 1998     

Regeneration of resistance and ion transport in rabbit corneal epithelium after induced surface cell exfoliation

Summary Exposure of the rabbit corneal surface to a 20-m digitonin-0.9% NaCl solution leads to permeabilization of the most superficial cells of the stratified epithelium. The devitalized cells exfoliate spontaneously from the corneal surface. Detergent exposure limited to 4–8 min leads to permeabilization and rapid exfoliation of a monolayer of surface cells. Consistent with the presence of the epithelial paracellular permeability barrier in this cell layer, their permeabilization results in complete loss of transepithelial resistance (R _t ). Within minutes after detergent removal an initial recovery ofR _t can be noticed indicating generation of a new paracellular permeability barrier by the viable subsurface cells. This recovery proceeds rapidly andR _t reaches within 70 min a maximum equal to > 90% of the preexfoliation values (=2.43 k·cm²,n=22). TheR _t recovery is fully blocked in a reversible manner by 10 m dihydrocytochalasin B. The recovery is not affected by inhibition of protein synthesis with 5 m cycloheximide. When the ocular surface is treated again with digitonin the permeabilization and exfoliation of a monolayer of cells and loss ofR _t are repeated. After the second detergent exposure an initial recovery ofR _t occurs as before within minutes. However, the pace ofR _t recovery is much slower: 4–5 hr are required to reach a stable maximalR _t values amounting to about 73% of initial control. This recovery can be fully blocked by 5 m cycloheximide indicating that protein synthesis is required for generation of tight junctions by the second subcellular layer. With only a fraction ofR _t recovered, short-circuit currents amounting to, at least, 50% of control values and attributable in part to cell-to-tear movement of Cl^– through the apical surface can be measured. This suggests that apical-type Cl^– channels are either present in the apically facing membrane of subsurface cells or that they are rapidly inserted in it from preexisting intracellular pools immediately following the devitalization of the surface cells by digitonin.     

Regeneration of Gossypium hirsutum and G. barbadense from shoot apex tissues for transformation

A method of regenerating cotton plants from the shoot apical meristem of seedlings was developed for use with particle gun and Agrobacterium-mediated transformation. This method was developed to circumvent the problems of genotype restriction and chromosomal damage frequently encountered in cotton regeneration in tissue culture through somatic embryogenesis. In this procedure, the cells of the shoot meristem are targeted for transformation. Normal and fertile plants of Gossypium barbadense Pima S-6, and 19 cultivars of G. hirsutum were regenerated using this method. Shoot regeneration from these tissues was direct and relatively rapid. A MS based, hormone-free medium could be used with all the varieties tested.This project was funded by grants from Cotton Incorporated, Nisshinbo Industries, and a grant from the Texas Agricultural Experiment Station to RHS. Texas Agricultural Experiment Station Technical Article TA-25667.     

Regeneration of lost siphon tissues in the tellinacean bivalve Nuttallia olivacea

The inhalant siphon of the tellinacean bivalve Nuttallia olivacea is an important prey item for juvenile stone flounder Platichthys bicoloratus in estuaries in Japan. We examined quantitative siphon regeneration of N. olivacea in rearing experiments of siphon-removed bivalves (> 30 mm shell length) both in the laboratory and in their natural habitat. Under laboratory conditions, siphon-removed bivalves regenerated lost tissues quantitatively at 15 and 25 °C 1 mo after siphon removal, although regeneration was incomplete. A 3-mo caging experiment in the field showed that great regeneration occurred in siphon-removed bivalves. However, the siphon weight of removed bivalves was significantly smaller than that of non-amputated bivalves, suggesting the incomplete regeneration. In a 1-mo caging experiment, bivalves that had approximately 15% of their siphons amputated were selected at some intervals to illustrate the quantitative regeneration process. Estimated daily siphon production was remarkably high only a few days after amputation. It decreased greatly thereafter, but regeneration was not completed within 30 d. These results indicate that bivalves regenerate siphons rapidly just after losing siphon tissues and then regeneration is slowed down before it is completed.     

Regeneration of radiation-damaged digestive tissues in juvenile Pacific oysters (Crassostrea gigas)

Juvenile Pacific oysters, Crassostrea gigas, were irradiated with 16 and 40 krad and their tissues examined histologically. Degenerative syndromes and tissue regeneration processes were determined for the stomach, gut, collecting ducts, and digestive tubules. Following degeneration, tissue regeneration was observed in the digestive tissues of most oysters exposed to 16 krad and in a limited number exposed to 40 krad. Regeneration was first observed in the digestive tubules and subsequently in the stomach, gut, and collecting ducts. Cellular repopulation of the digestive tubules involved epithelialization with large, undifferentiated crypt cells which then differentiated into functional secretory and absorptive cells. Regeneration in the stomach, gut, and collecting ducts was initiated by proliferative islands of small basophilic cells. Mitotic division of those cells and their subsequent differentiation into functional epithelial cells resulted in the rapid restoration and apparent recovery of the affected tissues. The results of these studies indicate that radioresistance of juvenile C. gigas may in part be due to the remarkably efficient regenerative mechanisms involved in replacing injured or lost digestive tissues.     

Keratocyte phenotype mediates proteoglycan structure: a role for fibroblasts in corneal fibrosis

In pathological corneas, accumulation of fibrotic extracellular matrix is characterized by proteoglycans with altered glycosaminoglycans that contribute to the reduced transparency of scarred tissue. During wound healing, keratocytes in the corneal stroma transdifferentiate into fibroblasts and myofibroblasts. In this study, molecular markers were developed to identify keratocyte, fibroblast, and myofibroblast phenotypes in primary cultures of corneal stromal cells and the structure of glycosaminoglycans secreted by these cells was characterized. Quiescent primary keratocytes expressed abundant protein and mRNA for keratocan and aldehyde dehydrogenase class 3 and secreted proteoglycans containing macromolecular keratan sulfate. Expression of these marker compounds was reduced in fibroblasts and also in transforming growth factor-beta-induced myofibroblasts, which expressed high levels of alpha-smooth muscle actin, biglycan, and the extra domain A (EDA or EIIIA) form of cellular fibronectin. Collagen types I and III mRNAs were elevated in both fibroblasts and in myofibroblasts. Expression of these molecular markers clearly distinguishes the phenotypic states of stromal cells in vitro. Glycosaminoglycans secreted by fibroblasts and myofibroblasts were qualitatively similar to and differed from those of keratocytes. Chondroitin/dermatan sulfate abundance, chain length, and sulfation were increased as keratocytes became fibroblasts and myofibroblasts. Fluorophore-assisted carbohydrate electrophoresis analysis demonstrated increased N-acetylgalactosamine sulfation at both 4- and 6-carbons. Hyaluronan, absent in keratocytes, was secreted by fibroblasts and myofibroblasts. Keratan sulfate biosynthesis, chain length, and sulfation were significantly reduced in both fibroblasts and myofibroblasts. The qualitatively similar expression of glycosaminoglycans shared by fibroblasts and myofibroblasts suggests a role for fibroblasts in deposition of non-transparent fibrotic tissue in pathological corneas.     

Mesenchymal cells emerge as primary contributors to fibrosis in multiple tissues

A longstanding controversy exists regarding the cellular origin of myofibroblasts in tissue fibrosis. A recent study by Hung and colleagues (Am J Respir Crit Care Med 188(7):820–830, 2013) used genetic fate mapping of FoxD1 embryonic progenitor cells to show a major and direct contribution of mesenchymal cells to fibrogenesis in the lung. Future studies using FoxD1-specific inducible knockout models of pro-fibrotic genes such as CCN2 will be valuable for determining anti-fibrotic drug targets. The emergence of pericyte-like myofibroblast precursors also raises the question of whether mesenchymal stem cells in various niches contribute to fibrotic responses throughout the body.     

Activation of chloride secretion in cystic fibrosis cells and tissues by the substituted imidazole SRI 2931

Recent interest in nucleotides and related agents as part of clinical trials in cystic fibrosis (CF) therapy have elicited efforts to identify novel compounds capable of activating transepithelial chloride (Cl(-)) transport in CF cells and tissues. From a library of nucleosides, bases, and other substituted heterocycles, 341 compounds were screened for their ability to activate anion transport in CF cells grown on permeable supports. One compound, SRI 2931, was found to confer prolonged and potent activity when administered to the apical surfaces of CF pancreatic epithelial cells, primary CF nasal epithelial cells, non-CF human colonic epithelial cells, and intact tissue taken from mouse models for CF. Concentrations of SRI 2931 (20 microM), which activated Cl(-) transport, had minimal effect on cell proliferation. SRI 2931 was not calcium (Ca(2+)) or cAMP dependent, suggesting important differences from conventional chloride secretagogues. The compound selectively released ATP from the apical, but not basolateral, surfaces of CF cells grown on permeable supports. The magnitude, longevity, and mechanism of action of the response provide a tool for dissecting pathways of epithelial ATP extracellular signaling and Cl(-) permeability.     

Kallikrein and amylase contents in tissues from a mutant mouse model for human cystic fibrosis

Kallikrein and amylase activities are decreased in the pancreas and salivary glands from $\text{cri/cri}$ homozygote mutant mice. Kallikrein is decreased in the $\text{cri/cri}$ kidney too. With reference to nucleic acid concentrations there is no difference between control and mutant mice. The previously described electrolyte abnormalities of the cribriform degeneration $\text{(cri)}$ mutant mouse, could be due to the abnormal activity of the kallikrein-kinin system on the transport mechanism of tubular cells in the organs mentioned. These findings represent a new step on our efforts to develop a useful animal model for human cystic fibrosis research.     

Repair in avascular tissues: fibrosis in the transparent structures of the eye and thrombospondin 1.

Wound repair is a process which is normally dependent on the vasculature of the damaged tissue. However, the transparent structures of the eye (e.g. central cornea, lens, vitreous) are avascular and yet are still subject to repair and fibrosis. Moreover, the resulting ophthalmic scars often remain avascular. Since this type of ocular scarring may result in blindness, it is the subject of intense research. An aspect of avascular ophthalmic fibrosis which has attracted attention is the question concerning early wound healing components that are usually derived from blood constituents. One such molecule is the glycoprotein thrombospondin 1. Thrombospondin 1 is thought to be a key regulator of cell behaviour in early wound repair and appears to be derived totally from platelet alpha-granules during repair of incisional skin wounds. It has been shown that the ocular cells involved in avascular repair processes, and which are thus responsible for healing in the absence of platelet-derived thrombospondin 1, are capable of synthesizing the protein themselves. It is suggested that cells involved in ophthalmic repair processes produce thrombospondin 1 in the absence of the platelet-derived molecule. Local synthesis of thrombospondin 1 may represent a therapeutic target in the management of ophthalmic fibrosis.     

细胞因子与肝脏再生

肝脏再生是肝在受到损伤时产生的重要反应,该从细胞因子的角度,阐述了细胞因子和肝脏再生之间的关系。     

The effect of digestion with keratanase (Pseudomonas sp.) on certain histochemical reactions for glycosaminoglycans in cartilaginous and corneal tissues

Summary The effect of digestion with keratanase (Pseudomonas sp.) on the Alcian Blue (AB) pH 1.0, pH 2.5, Aldehyde Fuchsin, high iron diamine, low iron diamine and dialysed iron-ferrocyanide reactions has been tested in the costal and ear cartilage tissues of the rabbit and corneal tissues of the rat and rabbit. The effect of digestion with chondroitinases ABC and AC on the same reactions was examined in the same tissues for comparison. Digestion with keratanase diminished the intensity of all the reactions in the cartilage tissues to a variable extent; however, the diminutions in intensity of the reactions appeared to be less marked as compared with those following digestion with two chondroitinases. In the corneal stroma, all the reactions were markedly reduced in intensity following digestion with keratanase. In contrast, these reactions were only slightly or moderately diminished in intensity by digestion with the two chondroitinases. As glycosaminoglycans are known to be present in cartilage and corneal tissues and the substrate specificities of the three enzymes used are now well established, the present results are consistent with the concept that keratanase specifically degrades and releases keratan sulphates involved in the tissues.     

ALR and Liver Regeneration

Liver possesses the capacity to restore its tissue mass and attain optimal volume in response to physical, infectious and toxic injury. The extraordinary ability of liver to regenerate is the effect of cross-talk between growth factors, cytokines, matrix components and many other factors. In this review we present recent findings and existing information about mechanisms that regulate liver growth, paying attention to augmenter of liver regeneration.