WOUND HEALING AND COLLAGEN FORMATION : IV. Distortion of Ribosomal Patterns of Fibroblasts in Scurvy

The changes in scorbutic wounds following the administration of ascorbic acid have been investigated using the techniques of electron microscopy, histochemistry, and autoradioggraphy. Particular attention has been paid to the changes seen in the endoplasmic reticulum of the fibroblasts and to the identity of the extracellular filamentous material characteristic of scorbutic wounds. Seven-day-old wounds in scorbutic guinea pigs were examined prior to and from one to 72 hours following the administration of vitamin C. Fibroblasts from wounds of normal animals demonstrate a characteristic configuration of the ribosomes of the endoplasmic reticulum which is suggested to be analogous to polyribosomes described in cells synthesizing protein such as the reticulocyte. Tangential views of the membranes of the ergastoplasm show the ribosomes to be grouped in paired rows which take both straight and curved paths. This configuration is lost in scurvy and can be seen to begin to reappear as early as 4 hours after giving ascorbic acid. With increasing time, the morphology of the ribosomal aggregates approximates that seen in normal cells, so that by 24 hours their reorientation is complete. It is suggested that one of the disturbances in scurvy may relate to an alteration either in messenger RNA, in the ability of the ribosomes to relate to the messenger, or in the membranes of the ergastoplasm. In addition, the lack of formation of hydroxyamino acids necessary for completing collagen synthesis may be related to the architecture of the ribosomal aggregates. Extracellular collagen fibrils appear concomitant with the restoration of ribosomal and ergastoplasmic morphology as early as 12 hours after administration of ascorbic acid, with complete disappearance of the scorbutic extracellular material within 24 hours. Observations of this scorbutic material do not support the concept that it is a collagen precursor.     

Influence of ascorbic acid on ribosomal patterns and collagen biosynthesis in healing wounds of scorbutic guinea pigs

Scorbutic guinea pigs were wounded and the influence of administering ascorbic acid 6 days later was studied with respect to cellular morphology, ribosomal distribution and protein synthesis. Electron-microscopic studies revealed that the dilated endoplasmic reticulum observed in the fibroblasts of scorbutic wound tissue had reverted to a normal configuration 24h after intraperitoneal injection of 100mg of ascorbate. Quantitative determination of the distribution of free and membrane-bound ribosomes indicated a significant increase in membrane-bound ribosomes in wound tissue from ascorbate-supplemented (recovery) animals. Sucrose-density-gradient centrifugation indicated a significant increase in the proportion of large membrane-bound polyribosomes in the range 300-350S and a concomitant decrease in 80S monoribosomes in the ribosome sedimentation profile of recovery tissue. Determination of the synthesis of non-diffusible [(3)H]hydroxyproline in scorbutic and recovery wounds showed a 3-4-fold stimulation in peptidyl-proline hydroxylation in recovery tissues. Studies carried out in which scorbutic and recovery tissues were incubated with [(14)C]leucine indicated that general protein synthesis, as measured by (14)C incorporated into non-diffusible material/mug of DNA, was unaltered by ascorbate supplementation. Similar studies of [(3)H]proline incorporation suggested that in recovery tissues there was a small but significant increase in [(3)H]proline incorporated/mug of DNA, which probably represents an increase in protocollagen synthesis. This observation correlates well with the increase seen in recovery tissues of large polyribosomes on which collagen precursor polypeptides are known to be synthesized. Preliminary characterization of the repair collagen synthesized by recovery animals showed it to be a typical Type I collagen having the chain composition (alpha(1))(2)alpha(2). The extent of glycosylation of the hydroxylysine of the newly synthesized collagen was greater than that reported for either normal guinea-pig dermal collagen or dermal scar collagen.     

Decreased extracellular matrix production in scurvy involves a humoral factor other than ascorbate

Our recent studies suggested that decreased collagen synthesis in bone and cartilage of scorbutic guinea pigs was not related to ascorbate-dependent proline hydroxylation. The decrease paralleled scurvy-induced weight loss and reduced proteoglycan synthesis. Those results led us to propose that the effects of ascorbate deficiency on extracellular matrix synthesis were caused by changes in humoral factors similar to those that occur in fasting. Here we present evidence for this proposal. Exposure of chick embryo chondrocytes to scorbutic guinea pig serum, in the presence of ascorbate, led to effects on extracellular matrix synthesis similar to those seen in scorbutic animals. The rates of collagen and proteoglycan synthesis were reduced to approximately 30-50% of the levels in cells cultured in normal guinea pig serum plus ascorbate, but proline hydroxylation and procollagen secretion were unaffected. Similar results were obtained with serum from fasted guinea pigs supplemented in vivo with ascorbate. The growth rate of the chondrocytes was not significantly affected by scorbutic guinea pig serum.     

Studies in vivo on the biosynthesis of collagen and elastin in ascorbic acid-deficient guinea pigs

1. After the administration of labelled proline to guinea pigs deprived of ascorbic acid for 15 days, the dorsal skin was examined 5 days later in an attempt to detect the presence of hydroxyproline-deficient collagen (protocollagen). The extent of incorporation of proline into skin collagens indicated a severe impairment of collagen synthesis. 2. A comparison of proline and hydroxyproline specific radioactivities in diffusible peptides obtained by treatment with collagenase of either purified skin collagens or direct hot-trichloroacetic acid extracts of skin failed to indicate the presence of protocollagen. Possible reasons for this are discussed. 3. The incorporation results did not indicate an inability of normal collagen, i.e. collagen hydroxylated to the normal degree, to cross-link in scurvy. 4. Incorporation of labelled proline into aortic elastin isolated from the same animals did not indicate a decrease in elastin biosynthesis in ascorbic acid deficiency, beyond that attributable to the inanition accompanying the vitamin deficiency. The proline/hydroxyproline specific-radioactivity ratio in elastin from scorbutic guinea pigs was about 6:1 in contrast with the 1:1 ratio in control groups. It is concluded that the formation of elastin hydroxyproline was ascorbate-dependent and that a hydroxyproline-deficient elastin is formed and retained in scurvy. The formation of desmosines was unimpaired in scorbutic animals. 5. Studies with chick embryos confirmed the formation of elastin hydroxyproline from free proline. Incorporation of free hydroxyproline into elastin hydroxyproline was negligible. 6. Digestion of solubilized samples with collagenase indicated that the hydroxyproline in guinea-pig aortic elastin preparations was not derived from contamination by collagen. It is suggested that most if not all of the hydroxyproline in the guinea pig elastin preparations investigated can be considered an integral part of the elastin molecule.     

WOUND HEALING AND COLLAGEN FORMATION : II. Fine Structure in Experimental Scurvy

The sequence encountered in healing skin wounds in scorbutic guinea pigs has been examined by methods of light and electron microscopy. Linear incisions in the skin of female guinea pigs fed a scorbutigenic diet were allowed to heal. The wounds were removed for examination at 1, 3, 5, 9, and 14 days after wounding. The fibroblasts of the scorbutic wounds differ from those of the controls in three major aspects. First, little collagen is present within the intercellular spaces, although small groups of individual collagen fibrils can be found adjacent to some of the fibroblasts; in addition, large amounts of somewhat fibrillar, poorly structured, dense matter are present throughout the extracellular regions. The second alteration consists of large aggregates of intracytoplasmic lipid deposits present within the majority of the fibroblasts. Third, the endoplasmic reticulum of the fibroblasts is altered in form from that of the controls. The profiles of the cisternae are round, non-continuous within the plane of section, and are less extensively developed than in the controls. An increased macrophagic activity of the histiocytes is also described. These changes are discussed in light of the available biochemical data associated with this abnormality of protein synthesis.     

Coordinate regulation of collagen and proteoglycan synthesis in costal cartilage of scorbutic and acutely fasted, vitamin C-supplemented guinea pigs

The effects of ascorbic acid deficiency and acute fasting (with ascorbate supplementation) on the synthesis of collagen and proteoglycan in costal cartilages from young guinea pigs was determined by in vitro labeling of these components with radioactive proline and sulfate, respectively. Both parameters were coordinately decreased by the second week on a vitamin C-free diet, with a continued decline to 20-30% of control values by the fourth week. These effects were quite specific, since incorporation of proline into noncollagenous protein was reduced by only 30% after 4 weeks on the deficient diet. The time course of the decrease in collagen and proteoglycan synthesis paralleled the loss of body weight induced by ascorbate deficiency. Hydroxylation of proline in collagen synthesized by scorbutic costal cartilage was reduced to about 60% of normal relatively early, and remained at that level thereafter. Neither collagen nor proteoglycan synthesis was returned to normal by the addition of ascorbate (0.2 mM) to cartilage in vitro. Administration of a single dose of ascorbate to scorbutic guinea pigs increased liver ascorbate and restored proline hydroxylation to normal levels by 24 h, but failed to increase the synthesis of collagen or proteoglycan. Synthesis of both extracellular matrix components was restored to control levels after four daily doses of ascorbate. A 96-h total fast, with ascorbate supplementation, produced rates of weight loss and decreases in the synthesis of these two components similar to those produced by acute scurvy. There was a linear correlation between changes in collagen and proteoglycan synthesis and changes in body weight during acute fasting, scurvy, and its reversal. These results suggest that it is the fasting state induced by ascorbate deficiency, rather than a direct action of the vitamin in either of these two biosynthetic pathways, which is the primary regulatory factor.     

Studies in vivo on the biosynthesis of collagen and elastin in ascorbic acid-deficient guinea pigs. Evidence for the formation and degradation of a partially hydroxylated collagen

1. After the administration of l-[G-(3)H]proline to guinea pigs deprived of ascorbic acid for increasing periods of time, the specific radioactivities of proline and hydroxyproline in skin collagen and aortic elastin were determined at various time-intervals after administration of the labelled compound with a view to studying the formation and degradation of collagen and elastin both deficient in hydroxyproline. 2. As judged from the incorporation of radioactivity into elastin proline, elastin synthesis was not decreased in the ascorbic acid-deficient animals. There was however, a rapid decline in the specific radioactivity of elastin hydroxyproline. The proline/hydroxyproline specific-radioactivity ratio was approx. 1.5:1 after 6 days and 20:1 after 12 days of ascorbic acid deprivation, in contrast with the ratio of 1:1 in controls. The results suggested that the effect of ascorbic acid deficiency on elastin biosynthesis could be regarded as simply an elimination of hydroxylation of elastin proline with the formation and retention of a polymer increasingly deficient in hydroxyproline. 3. Collagen proline and hydroxyproline specific radioactivities were derived from material that was soluble in hot trichloroacetic acid, non-diffusible and collagenase-degradable. In contrast with elastin, there was a rapid decline in the specific radioactivity of proline as well as hydroxyproline in collagen from the ascorbic acid-deficient animals. However, the proline/hydroxyproline specific-radioactivity ratio in all samples from scorbutic animals was consistently slightly above 1:1. The results suggest the appearance in place of collagen, but in rapidly diminishing amounts, of a partially hydroxylated collagen in which the degree of hydroxylation may be decreased only by approx. 10%. 4. Incorporation of radioactivity into the diffusible hydroxyproline in skin remained relatively high despite the rapid decline in the incorporation of radioactivity into collagen. This observation is interpreted as indicative of an increasing degree of degradation of partially hydroxylated collagen to diffusible peptides. An alternative explanation might be that partially hydroxylated peptides are released to an increasing extent from ribosomes before they attain a length at least sufficient to render them non-diffusible. In either case it implies the accumulation in scurvy of low-molecular-weight peptides enriched in proline and deficient in hydroxyproline and could explain the failure to accumulate a high-molecular-weight collagen deficient in hydroxyproline. 5. It is thought, however, that, in addition, an inhibition of ribosomal amino acid incorporation leading to decreased synthesis of partially hydroxylated collagen may also occur, perhaps secondarily to impaired hydroxylation.     

Beta-hydroxyaspartic acid in vitamin K-dependent plasma proteins from scorbutic and warfarin-treated guinea pigs

beta- Hydroxyaspartic acid is a rare amino acid, present in all vitamin K-dependent plasma proteins except prothrombin, and is formed by a post-translational hydroxylation of aspartic acid. We have now investigated whether this hydroxylation, like that of proline in collagen, is vitamin C-dependent. The vitamin K-dependent plasma proteins were isolated from normal and scorbutic guinea pig plasma by barium citrate adsorption and the beta- hydroxyaspartic acid content was determined. Compared with normal animals, scorbutic animals showed no significant reduction of beta- hydroxyaspartic acid content. In warfarin-treated animals there was a decreased content of both beta- hydroxyaspartic acid and gamma-carboxyglutamic acid in the barium citrate adsorbed fraction. It was concluded that the post-translational hydroxylation of aspartic acid is unlikely to be vitamin C-dependent.     

WOUND HEALING AND COLLAGEN FORMATION : I. Sequential Changes in Components of Guinea Pig Skin Wounds Observed in the Electron Microscope

The regular sequence encountered in healing guinea pig skin wounds has been examined by methods of light and electron microscopy. Observations on cell populations, their fine structure, and fibril formation in the connective tissue have been made. Linear incisions in the skin of normal female guinea pigs weighing 300 to 350 grams were allowed to heal. The wounds were then excised, fixed with buffered 2 per cent osmium tetroxide, and postfixed in neutral buffered formalin, at 16 and 24 hours and at 3, 5, 9, and 14 days after wounding. They were then embedded in epoxy resin. In the inflammatory phase the exudate observed in the early wounds consists largely of polymorphonuclear neutrophilic leukocytes, macrophages, fibrin, and free extracellular organelles from the disrupted inflammatory cells. These organelles later appear in vacuoles in the cytoplasm of the macrophages. Fibroblasts first appear at 24 hours, and show extensive development and dilatation of the endoplasmic reticulum, which sometimes contains moderately dense flocculent material. In addition, these fibroblasts have enlarged mitochondria and condensations of filamentous material within the cytoplasm near the cell surface. Occasional myelin figures and moderately dense, 0.5 to 1.0 micron bodies are found within the cytoplasm of the early fibroblasts. Collagen fibrils are first seen at 3 days extracellularly near the cell surfaces. They appear at the later times in two populations of sizes. With increasing wound age the fibroblasts retain their morphology and the wounds decrease in cellularity concomitantly with the formation of increasing amounts of collagen. Several proposed mechanisms of collagen fibril formation are discussed in relation to the observed phenomena. The problem of correlating fibril diameter with the appearance of the periodic structure of collagen in relation to the minimal size fibril which would be anticipated to display this appearance is discussed.     

Scorbutic and fasted guinea pig sera contain an insulin-like growth factor I-reversible inhibitor of proteoglycan and collagen synthesis in chick embryo chondrocytes and adult human skin fibroblasts

Chick embryo chondrocytes cultured in sera from scorbutic and fasted guinea pigs exhibited decreases in collagen and proteoglycan production to about 30-50% of control values (I. Oyamada et al., 1988, Biochem. Biophys. Res. Commun. 152, 1490-1496). Here we show by pulse-chase labeling experiments that in the chondrocyte system, as in the cartilage of scorbutic and fasted guinea pigs, decreased incorporation of precursor into collagen was due to decreased synthesis rather than to increased degradation. There was a concomitant decrease in type II procollagen mRNA to about 32% of the control level. As in scorbutic cartilage, proteoglycan synthesis by chondrocytes in scorbutic serum was blocked at the stage of glycosaminoglycan chain initiation. Scorbutic and fasted guinea pig sera also caused a 50-60% decrease in the rates of collagen and proteoglycan synthesis in adult human skin fibroblasts, which synthesize mainly type I collagen. Decreased matrix synthesis in both cell types resulted from the presence of an inhibitor in scorbutic and fasted sera. Elevated cortisol levels in these sera were not responsible for inhibition, as determined by the addition of dexamethasone to chondrocytes cultured in normal serum. Insulin-like growth factor I (IGF-I, 300-350 ng/ml) reversed the inhibition of extracellular matrix synthesis by scorbutic and fasted guinea pig sera in both cell types and prevented the decrease in type II procollagen mRNA in chondrocytes. Therefore, in addition to its established role in proteoglycan metabolism, IGF-I also regulates the synthesis of several collagen types. An increase in the circulating inhibitor of IGF-I action thus could lead to the negative regulation of collagen and cartilage proteoglycan synthesis that occurs in ascorbate-deficient and fasted guinea pigs.     

An electron microscopic autoradiographic study of proline incorporation by mouse lingual epithelium

Summary Mouse lingual epithelium incorporates significant amounts of L-proline-2, 3-H³ one hour after intraperitoneal injection of the tritiated amino acid. All viable cell strata incorporated approximately equal amounts of proline as assessed by autoradiographic techniques. Grain counts at 30 minutes, 1 hour, 4 hours and 24 hours, the four time periods studied, indicated a progressive incorporation of proline up to 4 hours following injection. Preferential incorporation of proline into any one cell structure or group of structures was not observed. Keratohyalin granules (KHG's) demonstrated incorporated proline; however, usually only one silver grain appeared over each granule, and, based on grain counts, the amount of proline incorporated by KHG's appeared slightly less than the general labeling observed in KHG-containing cells. This finding supports recent biochemical studies which have indicated a considerably lower proline content of keratohyalin than had previously been reported. Significant proline incorporation into the epithelial basal lamina was not observed during the 24 hours of this study. Thus, while recent recombination experiments have conclusively demonstrated that epithelial basal cells synthesize considerable quantities of basal lamina in a 24 hour period; it would appear that epithelial basal cells contribute little to a formed, intact basal lamina. This finding lends credence to the concept of a long basal lamina turnover time.Supported by Public Healths Service grants DE 02731, DE 03393The authors are grateful to Dr. John H. Lillie for his help in determining blood levels of proline-H³ and to Dr. V. C. Hascall for his advice on isotope selection. Mrs. K. Y. Y. Chen performed nearly all technical matters associated with this study, and made many of the original electron microscopic observations. Her assistance was invaluable.     

Self-assembly of collagen fibers. Influence of fibrillar alignment and decorin on mechanical properties.

Collagen is the primary structural element in extracellular matrices. In the form of fibers it acts to transmit forces, dissipate energy, and prevent premature mechanical failure in normal tissues. Deformation of collagen fibers involves molecular stretching and slippage, fibrillar slippage, and, ultimately, defibrillation. Our laboratory has developed a process for self-assembly of macroscopic collagen fibers that have structures and mechanical properties similar to rat tail tendon fibers. The purpose of this study is to determine the effects of subfibrillar orientation and decorin incorporation on the mechanical properties of collagen fibers. Self-assembled collagen fibers were stretched 0-50% before cross-linking and then characterized by microscopy and mechanical testing. Results of these studies indicate that fibrillar orientation, packing, and ultimate tensile strength can be increased by stretching. In addition, it is shown that decorin incorporation increases ultimate tensile strength of uncross-linked fibers. Based on the observed results it is hypothesized that decorin facilitates fibrillar slippage during deformation and thereby improves the tensile properties of collagen fibers.     

Mechanism for the decreased biosynthesis of cartilage proteoglycan in the scorbutic guinea pig

Our previous work showed that vitamin C deficiency caused about a 70-80% decrease in the incorporation of [35S]sulfate into proteoglycan of guinea pig costal cartilage, coordinately with a decrease in collagen synthesis (Bird, T. A., Spanheimer, R. G., and Peterkofsky, B. (1986) Arch. Biochem. Biophys. 246, 42-51). We examined the mechanism for decreased proteoglycan synthesis by labeling normal and scorbutic cartilage in vitro with radioactive precursors. Proteoglycan monomers from scorbutic tissue were of a slightly smaller average hydrodynamic size than normal but there was no difference in the size of the glycosaminoglycan chains isolated after papain digestion. The type of glycosaminoglycans synthesized and the degree of sulfation were unaffected as determined by chondroitinase ABC digestion and duel labeling with [35S]sulfate and [3H]glucosamine. Conversion of [3H]glucosamine to [3H]galactosamine also was unimpaired. There was about a 40% decrease in core protein synthesis, measured by [14C]serine incorporation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Nevertheless, decreased incorporation of [35S]sulfate into scorbutic tissue persisted in the presence of p-nitrophenyl-beta-D-xyloside and cycloheximide, which indicated that the site of the scorbutic defect was beyond core protein synthesis and xylosylation. Galactosyltransferase activity in scorbutic cartilage decreased to about one-third the levels in control samples in parallel with the decreases in proteoglycan and collagen synthesis. Our results suggest that the step catalyzed by this enzyme activity, the addition of galactose to xylose prior to chondroitin sulfate chain elongation, is the major site of the scorbutic defect in proteoglycan synthesis. Decreased enzyme activity may be related to increased cortisol levels in scorbutic serum.     

An autoradiographic and electron microscopic study of collagen synthesis in differentiating cartilage

Summary The synthesis of the proline-rich collagen component of cartilage matrix has been studied by autoradiography using both the light and electron microscope. Amblystoma maculatum larvae had their forelimbs amputated, were allowed to regenerate for 12–15 days, and then injected intraperitoneally with tritiated proline. The animals were fixed at various times (1 min. to 28 days) after the injection and sections of the developing limbs were coated for autoradiography by dipping in Ilford L 4 or Gevaert 3.07 emulsion. The sequential labeling of the organelles of the cartilage cell which occurred is illustrated in light and electron micrographs. Radioactive products first appeared in the ergastoplasm and were associated with the cisternae of the endoplasmic reticulum. Twenty to thirty minutes after the injection, labeled material began to appear in the Golgi zone. There, the newly synthesized protein accumulated within large vacuoles. The fibrillar material within the vacuoles may represent collagen and the more amorphous material, mucoprotein. The vacuoles subsequently (2 hrs. later) discharge their labeled contents into the extracellular space. The secreted protein is probably soluble collagen (tropocollagen) for it diffuses readily through the matrix to polymerize into striated collagen fibrils some distance from the cell. These findings contradict some widely held opinions that the fibrillar component of the matrix arises by excortication and appositional growth of fibrils originating from the ectoplasm of chondrocytes. It seems reasonable to conclude that the secretory pathway by which extracellular proteins are produced in cartilage is analogous to that suggested for epithelial gland cells.Supported by grants CA 05196-04S1 and GM-K3-13, 979-C1-A from the United States Public Health Service.The results reported in this paper were presented at the second annual meeting of the American Society for Cell Biology, November 6, 1962.     

Effects of adrenalectomy and excess hydrocortisone on the incorporation of 35S-labelled cysteine in the hypothalamic-neurohypophyseal neurosecretory system of the rat

Summary The incorporation of ³⁵S-labelled cysteine in the hypothalamic-neurohypophyseal system was studied in normal and adrenalectomized rats and in rats treated with excess hydrocortisone. Labelled cysteine was intraperitoneally administered and grain counts were made of autoradiographs produced from sections of the supraoptic and paraventricular nucleus, median eminence and neurohypophysis of animals killed 45 min., 4 hours and 24 hours after administration of the labelled substance. On the whole, lower incorporation levels of the label were noted in the adrenalectomized rats, compared with the controls. In the rats treated with excess hydrocortisone, the grain counts at 45 min and 4 hours after injection were higher and those at 24 hours were lower than those of the controls.The findings are discussed, among other things, in terms of rate of uptake vs. time and related to previous reports on the cysteine uptake and neurosecretory activity of the hypothalamic-neurosecretory sytem.This study was financially supported by the Sigrid Jusélius Foundation, Helsinki, Finland and the National Research Council for Medical Sciences, Finland.     

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) does not destroy nigrostriatal neurons in the scorbutic guinea pig

Guinea pigs were injected subcutaneously with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in maximal tolerated doses (8 mg/kg, once daily) for 10 or 15 days. No neurological effects were noted, other than sedation and hypotonia lasting a few hours after each injection, either in animals maintained on normal diet or in animals fed an ascorbate-deficient diet and rendered severely scorbutic. Subsequent chemical analyses of the striatum showed no evidence of lasting damage to nigrostriatal dopaminergic neurons in MPTP treated guinea pigs on normal diet, and minimal evidence of permanent damage to these neurons in scorbutic animals. MPTP was undetectable in the urine of MPTP-treated animals, although a metabolite, presumably 1-methyl-4-phenylpyridinium ion (MPP+) was regularly present in urine. The relative lack of neurotoxicity of MPTP in the guinea pig remains unexplained. This species clearly is not a suitable small animal for MPTP-induced parkinsonism.     

Complement component C1q is sensitive to acute vitamin C defficiency in guinea pigs

In acutely scorbutic guinea pigs, where interstitial collagen synthesis is markedly impaired, there was no significant reduction in total complement component C1 activity measured by a functional assay, and no significant reduction in the ratio of protein-bound hydroxyproline to protein-bound proline or to total serum protein, in comparison with pair-fed controls. There was a moderate increase in non-protein-bound hydroxyproline in the serum of the deficient animals.These result suggest that component C1q is largely resistant to the effects of severe acute scurvy, adn that some hydroxyproline-containing proteins may respond differently others, during vitamin C deficiency.     

Effect of hydrocortisone on skin development in the chick embryo: ultrastructural, immunohistological, and biochemical analysis

The effect of hydrocortisone on the development of dorsal skin was analyzed in the chick embryo by (1) transmission electron microscopy, (2) indirect immunofluorescence histology of extracellular matrix components (collagen types I, III, and IV; fibronectin; and laminin), and (3) quantitative determination of collagen content and proline incorporation, between administration of the drug at 6 or 6.5 days and final retrieval of skin pieces at 11 days of incubation. Treatment caused the formation of featherless skin areas which exhibited an early maturation of the epidermis, a uniform distribution of interstitial collagen and rarefaction of fibronectin in the dermal extracellular matrix, and a significant increase of collagen content and proline incorporation in collagen noncollagen proteins, characterized by an increased hydroxyproline-to-proline ratio. The distribution of type IV collagen and of laminin was unchanged. The absence of feather formation in hydrocortisone-induced apteria is interpreted as resulting primarily from an early extinction of epidermal morphogenetic competence, and secondarily from modifications in the amount and distribution of extracellular matrix components in the dermis.     

SPARC regulates collagen interaction with cardiac fibroblast cell surfaces

Cardiac tissue from mice that do not express secreted protein acidic and rich in cysteine (SPARC) have reduced amounts of insoluble collagen content at baseline and in response to pressure overload hypertrophy compared with wild-type (WT) mice. However, the cellular mechanism by which SPARC affects myocardial collagen is not clearly defined. Although expression of SPARC by cardiac myocytes has been detected in vitro, immunohistochemistry of hearts demonstrated SPARC staining primarily associated with interstitial fibroblastic cells. Primary cardiac fibroblasts isolated from SPARC-null and WT mice were assayed for collagen I synthesis by [(3)H]proline incorporation into procollagen and by immunoblot analysis of procollagen processing. Bacterial collagenase was used to discern intracellular from extracellular forms of collagen I. Increased amounts of collagen I were found associated with SPARC-null versus WT cells, and the proportion of total collagen I detected on SPARC-null fibroblasts without propeptides [collagen-α(1)(I)] was higher than in WT cells. In addition, the amount of total collagen sensitive to collagenase digestion (extracellular) was greater in SPARC-null cells than in WT cells, indicating an increase in cell surface-associated collagen in the absence of SPARC. Furthermore, higher levels of collagen type V, a fibrillar collagen implicated in collagen fibril initiation, were found in SPARC-null fibroblasts. The absence of SPARC did not result in significant differences in proliferation or in decreased production of procollagen I by cardiac fibroblasts. We conclude that SPARC regulates collagen in the heart by modulating procollagen processing and interactions with fibroblast cell surfaces. These results are consistent with decreased levels of interstitial collagen in the hearts of SPARC-null mice being due primarily to inefficient collagen deposition into the extracellular matrix rather than to differences in collagen production.