Type I and type III collagen content of healing wounds in fetal and adult rats

Full-thickness, dermal wounds were surgically created on the dorsa of fetal rats on the 17th day of gestation. The granulation tissue which developed after 2 days (19 days of gestation) was harvested from six to nine animals and pooled and the collagen was extracted with 0.5 M acetic acid and acetic acid plus pepsin. The ratio of type III:type I collagen was estimated from densitometer scans of electrophoretically separated alpha-chains. Full-thickness (to fascia depth) wounds were also produced on the dorsa of adult rats and granulation tissue which had developed for different periods of time up to 30 days was excised. Relative proportions of type III and type I collagen were assessed in normal and granulation tissues taken from the adult rats. Both fetal and adult granulation tissues have elevated type III collagen content but normal fetal tissue has a much higher content of type III than does normal adult tissue.     

Processing of types I and III procollagen in Ehlers-Danlos syndrome type VII.

The processing of types I and III procollagen was studied in skin fibroblast cultures from type VII A and B of the Ehlers-Danlos syndrome [EDS] and age-matched controls. Synthesis of collagenous proteins was significantly increased in EDS type VII B, and the activities of prolyl-4-hydroxylase and galactosylhydroxylysyl glucosyltransferase were slightly increased in these cell lines, reflecting increased biosynthesis of collagen. The synthesis of collagenous proteins was close to normal in EDS type VII A cells. The synthesis of type III procollagen per cell was increased, as also was the ratio of immunoreactive type III procollagen to total collagen production. The activity of type I procollagen amino-terminal proteinase was decreased in skin fibroblasts of type VII A and normal in those of type VII B relative to cell protein or DNA. Type III amino-terminal proteinase activity was of a level found in normal cells when expressed relative to the protein or DNA, and the release of type III amino-terminal propeptides was nevertheless not disturbed in these EDS type VII cell cultures. The results show that only the conversion of type I procollagen is defective in EDS type VII, and no general defect in procollagen processing can be found in EDS type VII as has been suggested in the case of dermatosparaxis, a connective tissue disorder in animals caused by disturbed procollagen conversion.     

Formation of liquid crystal structures in the tissue fluid in wound healing during periodic irradiation with helium-neon laser

Rabbit tissue fluid at different stages of wound healing was studied. Interaction between the formation of liquid crystalline fractions and the course of healing was revealed both under natural conditions and under periodic laser irradiation of the wound.     

Dynamics of wound healing of skin wounds produced by laser and scalpel in experiments

In this study in 90 rats the measured rate of healing, pO2 in sites of postoperative scar and histological specimen of identical excisional wounds produced by CO2-laser, scalpel and combination are compared. The data of healing laser wounds via scalpel ones show slow laser wound healing. We explain, this by inhibition of the inflammation phase due to diminishing macrophages migration into the wound. pO2 data in postoperative scar after laser and scalpel wounds show that laser thermal alteration does not influence CO2 regimen in the scar.     

Thrombospondin 2 levels are increased in aged mice: consequences for cutaneous wound healing and angiogenesis.

The inhibitor of angiogenesis, thrombospondin 2 (TSP2), belongs to a group of matricellular proteins that are induced in response to injury and modulate the healing of dermal wounds. Thus, TSP-2-null mice display abnormal connective tissue architecture and increased angiogenesis in the dermis, and heal wounds at an accelerated rate. In this study, we report that the content of TSP2 is increased in the uninjured skin of aged mice. Furthermore, in primary dermal fibroblasts, TSP2 expression is increased both as a function of the age of the donor and days in culture. To determine the significance of the increased TSP2 in aged mice (two years or older), we performed full-thickness excisional wounds and compared their healing in aged and young (3-4 months) wild-type and TSP2-null mice. Gross morphological examination of wounds indicated that aged TSP2-null mice healed faster than their aged wild-type counterparts, but healing in aged mice was always sub-optimal in comparison to that in young animals. Surprisingly, despite the increase in TSP2, a potent inhibitor of angiogenesis, in wounds in aged mice, the vascular density of these wounds was not reduced in comparison to that in young animals. However, immunohistochemical analysis of healing wounds revealed a shift in the peak content of TSP2, from day 10 in young mice to day 14 or later in aged mice, and there was a corresponding delay in the expected increase in matrix metalloproteinase (MMP) 2 levels in aged TSP2-null mice. We suggest that the delay in expression of TSP2 and MMP2 in the wounds of aged mice could contribute to their impaired rate of wound healing.     

Demethylation of the type I procollagen genes in transformed fibroblasts treated with 5-azacytidine

Transformation of the human embryonic lung fibroblast line, WI-38, with simian virus 40 (SV40) results in inactivation of the type I procollagen genes. No type I collagen or procollagen mRNA is detected in these transformed cells, as determined by polyacrylamide gel electrophoresis. Analysis of the methylation patterns of these genes showed the type I procollagen genes to be hypermethylated at certain cytosine residues in the transformed cells. However, several of the cytosine residues were methylated in the normal cells where these genes are expressed. These methylation patterns can be altered by treatment of the cells with 5-azacytidine or 5-azadeoxycytidine, but without a resultant activation of the type I procollagen genes. These results show that demethylation alone is not sufficient for gene activation, but that other signals are also required.     

Enhanced healing of cutaneous wounds in rats using beads with positively charged surfaces.

The efficacy of electrical fields in soft-tissue repair is unclear. Materials with a charged surface provide a localized charged environment. We examined the effects of surface-charged particles in wound healing in rats with paired dorsal incisions with one side serving as a control. Tensiometry demonstrated that after 10 days, wounds with positively charged particles were 53 percent stronger (p less than 0.001) than controls (10 rats, 30 wound strips), whereas differences with negatively charged (6 rats, 15 strips) or uncharged beads (11 rats, 33 strips) were insignificant. Histologically, wounds with positively charged particles were characterized by large quantities of collagen-rich connective tissue and by prominent bead-associated giant cells. At 94 days, no differences in wound strength were noted. This method of creating charged local environments has potential clinical implications and may add insights into the behavior of cells in response to charged stimuli.     

Structure of cDNA clones coding for the entire prepro alpha 1 (III) chain of human type III procollagen. Differences in protein structure from type I procollagen and conservation of codon preferences.

Two overlapping cDNA clones that cover the complete length of the mRNA for human type III procollagen were characterized. The data provided about 2500 base pairs of sequence not previously defined for human type III procollagen. Two tripeptide sequences of -Gly-Xaa-Yaa- were identified that were not detected previously by amino acid sequencing of human type III collagen. The two additional tripeptide units, together with three previously detected, establish that the alpha 1 (III) chain is 15 amino acids longer than either the alpha 1 (I) or alpha 2 (I) chains of type I collagen. The additional tripeptide units made hydropathy plots of the N-terminal and C-terminal regions of type III collagen distinctly different from those of type I collagen. The data also demonstrated that human type III procollagen has the same third base preference in codons for glycine, proline and alanine that was previously found with human and chick type I procollagen. In addition, comparison of two cDNA clones from the same individual revealed a variation in structure in that the codon for amino acid 880 of the alpha 1 (III) chain was -CTT- for leucine in one clone and -TTT- for phenylalanine in the other.     

SPARC-thrombospondin-2-double-null mice exhibit enhanced cutaneous wound healing and increased fibrovascular invasion of subcutaneous polyvinyl alcohol sponges.

Secreted protein acidic and rich in cysteine (SPARC) and thrombospondin-2 (TSP-2) are structurally unrelated matricellular proteins that have important roles in cell-extracellular matrix (ECM) interactions and tissue repair. SPARC-null mice exhibit accelerated wound closure, and TSP-2-null mice show an overall enhancement in wound healing. To assess potential compensation of one protein for the other, we examined cutaneous wound healing and fibrovascular invasion of subcutaneous sponges in SPARC-TSP-2 (ST) double-null and wild-type (WT) mice. Epidermal closure of cutaneous wounds was found to occur significantly faster in ST-double-null mice, compared with WT animals: histological analysis of dermal wound repair revealed significantly more mature phases of healing at 1, 4, 7, 10, and 14 days after wounding, and electron microscopy showed disrupted ECM at 14 days in these mice. ST-double-null dermal fibroblasts displayed accelerated migration, relative to WT fibroblasts, in a wounding assay in vitro, as well as enhanced contraction of native collagen gels. Zymography indicated that fibroblasts from ST-double-null mice also produced higher levels of matrix metalloproteinase (MMP)-2. These data are consistent with the increased fibrovascular invasion of subcutaneous sponge implants seen in the double-null mice. The generally accelerated wound healing of ST-double-null mice reflects that described for the single-null animals. Importantly, the absence of both proteins results in elevated MMP-2 levels. SPARC and TSP-2 therefore perform similar functions in the regulation of cutaneous wound healing, but fine-tuning with respect to ECM production and remodeling could account for the enhanced response seen in ST-double-null mice.     

Coordinate regulation of the levels of type III and type I collagen mRNA in most but not all mouse fibroblasts

A mouse genomic clone was isolated by cross-hybridization with a DNA fragment which codes for the NH2-propeptide of chick alpha1(III) collagen. The region of cross-hybridization within the mouse clone was localized, its sequence determined, and an exon coding for the NH2-propeptide of mouse alpha1(III) collagen was identified. This DNA fragment hybridizes to an RNA species of approximately 5300 nucleotides, slightly larger than the major alpha2(I) collagen RNA species. The mouse type III collagen probe was used to examine the effect of transformation on alpha1(III) collagen RNA levels in mouse fibroblasts. The levels of type III and type I collagen mRNA levels were compared in control and sarcoma virus-transformed murine cell lines, as well as in NIH 3T3 cells transformed by members of the human ras oncogenes. The levels of type III RNA decreased about 10-15-fold in Moloney sarcoma virus-transformed cells and in a cell line transformed with a v-mos-containing plasmid, but showed only a 50% decrease in a Kirsten murine sarcoma virus-transformed BALB 3T3 cell line, and increased 4-fold in a Rous sarcoma virus (RSV)-transformed BALB 3T3 cell line. In contrast, the levels of alpha2(I) collagen mRNA are 8- to 10-fold lower in all these cell lines when compared to untransformed cells. NIH 3T3 cells transformed with two human ras oncogenes showed decreased levels of alpha2(I) and alpha1(III) mRNAs. In contrast to the RSV-transformed mouse cell line, RSV-transformed chick embryo fibroblasts contained much smaller amounts of type III RNA than control chick embryo fibroblasts. We conclude that the levels of alpha1(III) and alpha2(I) collagen mRNA are often but not necessarily coordinately regulated by transformation in mouse cells.     

Cleavage of type I and type II procollagens by type I/II procollagen N-proteinase. Correlation of kinetic constants with the predicted conformations of procollagen substrates

The kinetic constants were examined for the cleavage of several types of procollagen by type I/II procollagen N-proteinase. The Km values were essentially the same (0.2 microM) for chick type I procollagen, human type I procollagen, and chick type II procollagen. However, the Vmax values differed over a 14-fold range. As reported previously, the enzyme did not cleave denatured type I or II procollagen. Also, it did not cleave human type III procollagen which contains the same scissle -Pro-Gln- bond as the pro-alpha 1(I) chain of type I procollagen. To explain the observations, Chou-Fasman rules were used to compare the secondary structures of the cleavage sites in the procollagens. The results supported a previous suggestion (Helseth, D. L., Jr., Lechner, J. L., and Veis, A. (1979) Biopolymers 18, 3005-3014) that the region carboxyl-terminal to cleavage site in the pro-alpha 1(I) chain of type I procollagen was in a hairpin conformation consisting of a beta-sheet, beta-turn, and beta-sheet. In both chick and human type I procollagen, the hairpin loop in the pro-alpha 1(I) chain consisted of about 18 amino acids. The cleavage site itself was in a short alpha-helical structure of four or five amino acids. The pro-alpha 2(I) chains had a similar hairpin loop of about 14 amino acids and alpha-helix of four or five amino acids containing the cleavage site. Chick type II procollagen, which had the highest Vmax value, had a longer hairpin structure of 22 amino acids, and the cleavage site was in a longer alpha-helical domain of 10 amino acids. In contrast, type III procollagen had a random-coil conformation in the same region. The results help to explain the unusual substrate requirements of type I/II N-proteinase. They also help explain why mutations that produce in-frame deletions of amino acids 84 or more residues carboxyl-terminal to the cleavage site make the protein resistant to the enzyme.     

Construction and identification of a cDNA clone for human type II procollagen mRNA.

Double-stranded cDNA was constructed for poly(A)-containing RNA isolated from foetal human articular cartilage known to contain small amounts of pro alpha 1 (II) collagen mRNA. A 585 base pair PstI-EcoRI cDNA fragment was isolated and cloned into plasmid pBR322. A resulting recombinant plasmid pHCAR1 was shown to hybridize specifically to a 5.4 kilobase mRNA in cartilage but not in calvarial RNA. Definite identification of clone pHCAR1 was based on sequence analysis; marked homology with the corresponding chick gene and complete agreement with the human gene sequences available were observed.     

Synthesis of structurally unstable type III procollagen in patients with cerebral artery aneurysm.

The biosynthesis of collagen was studied in skin fibroblast cultures established from 11 patients with cerebral artery aneurysms. Six patients had familial subarachnoid hemorrhage (SAH), while five patients were considered as sporadic cases. The structural stability of the triple-helical medium procollagen was studied by measuring the thermal denaturation temperature (Tm) of type I and type III procollagen molecules. Structural instability of type III procollagen was demonstrated in two patients with familial SAH. The Tm of type III procollagen was 39.0 degrees C and 39.5 degrees C in two of the cell lines, while the control value was 40.3 degrees C. The stability of type I procollagen did not differ from that of the controls, and the main features of the biosynthesis of collagen were similar in the aneurysm patient cell lines and in the controls. The results suggest that a structural defect of type III procollagen may serve as an etiological factor in the formation of cerebral artery aneurysms.     

Low rate of procollagen conversion in dermatosparactic sheep fibroblasts is paralleled by increased synthesis of type I and type III collagens.

Dermatosparaxis is a genetic defect found in humans and animals, which affects the conversion of procollagen to collagen and aminopropeptides . Under cell culture conditions a reduction of the release of aminopropeptides is paralleled by an increased synthesis of collagens type I and III. This fact provides further evidence for a regulatory involvement of aminopropeptides in the biosynthesis of collagen.