Differential response to transforming growth factors-beta(1) and beta(2): connective tissue deposition in animal models.

The ability of transforming growth factor (TGF)-beta(1) and TGF-beta(2) to promote connective tissue deposition were compared in different animal models. A single subcutaneous injection of TGF-beta(2) in collagen/heparin gel carrier promoted markedly more extensive development of connective tissue than TGF-beta(1) at the site of injection in both neonatal and adult mice. Both TGF-beta(1) and TGF-beta(2) promoted deposition of dense and well-vascularized connective tissue matrix infiltrated with macrophages and fibroblasts. However, the results of immunohistochemical analyses suggested that TGF-beta(2) promoted an accumulation of more macrophages in the connective tissue than TGF-beta(1). Similar differences in the extent of connective tissue development were observed in neonatal mice when these factors were administered as a solution, without the collagen/heparin gel carrier. TGF-beta(2) was also more potent than TGF-beta(1) in domestic pigs. However, in guinea pigs, TGF-beta(1) promoted more extensive connective tissue development than TGF-beta(2). These results suggest that the differential connective tissue response to TGF-beta(1) and TGF-beta(2) is species dependent. However, the differences in the physical and chemical properties of these factors may account in part for the differential response as well.

Extravasation of macromolecules and possible trapping of transforming growth factor-beta in venous ulceration.

The pathogenesis of venous ulceration is thought to involve formation of pericapillary fibrin cuffs as a result of venous hypertension, and a recent hypothesis suggests that extravasated plasma proteins may bind or trap growth factors. We have compared the tissue distribution of fibrin cuffs, plasma proteins, procollagen, and transforming growth factors (TGF-beta 1 and TGF-beta 2) within venous ulcers and normally healing graft donor sites. In venous ulcers, the papillary dermis and the ulcer bed contained convoluted capillaries with phosphotungstic acid haematoxylin-positive pericapillary fibrin cuffs. By immunohistochemical staining, the cuffs were positive for actin, and contained massively redundant lamellae of basement membrane material which stained positive for type IV collagen. Extravasated factor XIIIa and alpha 2-macroglobulin were present within the fibrin cuffs. Increased numbers of type I procollagen positive fibroblasts, and increased TGF-beta 1 immunoreactivity were present within the fibrin cuffs, but not in the provisional matrix in the ulcer bed around the cuffs. In contrast, in normally healing graft donor sites, tortuous capillaries and fibrin cuffs were absent, factor XIIIa and alpha 1-macroglobulin were restricted to the lumina of vessels, and procollagen and TGF-beta immunoreactivity were present within the granulation tissue and adjacent dermal matrix at the wound margin. These observations suggest that growth factors critical in wound healing, such as TGF-beta, are present within venous ulcers, but are abnormally distributed. Their distribution within fibrin cuffs and co-localization with extravasated plasma proteins, particularly alpha 2-macroglobulin, which is a recognized scavenger molecule for TGF-beta and other growth factors, provides evidence for a possible 'trapping' of growth factors in venous ulcers.

Exogenous transforming growth factor-beta(2) enhances connective tissue formation in transforming growth factor-beta(1)-deficient, healing-impaired dermal wounds in mice.

Connective tissue formation is markedly reduced in full-thickness mouse dermal wounds that are covered with synthetic, adherent, moisture vapor-permeable membrane when compared with formation in similar but nonoccluded wounds. The transforming growth factors-beta (TGF-beta) are a family of multifunctional peptides thought to have a critical role in the regulation of development and tissue repair. Treatment with exogenous TGF-beta(1) stimulated connective tissue formation in wounds covered with synthetic, adherent, moisture vapor-permeable membrane but had no effect on air-exposed wounds, suggesting that the quantity of endogenous TGF-beta(1) in wounds covered with synthetic, adherent, moisture vapor-permeable membrane was less than that in air-exposed wounds. Immunolocalization studies with an anti-TGF-beta(1) antibody confirmed that wounds covered with synthetic, adherent, moisture vapor-permeable membrane demonstrated markedly reduced levels of endogenous extracellular, matrix-associated TGF-beta(1) as early as 24 hours after wounding. Immunoreactive TGF-beta(2) was not detected. These findings suggest that endogenous TGF-beta(1), but not TGF-beta(2), is required for normal connective tissue formation in this model and that impaired healing is associated with low levels of TGF-beta(1). Histologic analysis confirmed previous demonstrations that exogenous TGF-beta(2) stimulates enhanced cellularity and connective tissue formation. Immunolocalization showed that exogenous TGF-beta(2) stimulates increased expression of endogenous TGF-beta(1). Northern blot analysis revealed that TGF-beta(2) increased the expression of genes encoding the alpha(1)-chain of types I and III collagens and tissue inhibitor of metalloproteinase-1. These observations show that TGF-beta(2) acts through a variety of mechanisms to stimulate repair in healing-impaired wounds that are also deficient in endogenous TGF-beta(1), but they do not distinguish between direct effects and indirect effects mediated by induced TGF-beta(1).

Skin distribution and differential expression of transforming growth factor beta 1 and beta 2.

Transforming growth factor beta (TGF-beta) 1 and 2 have both become increasingly important in cutaneous biology, but their expression and distribution in human skin are not entirely clear. In this report, normal forearm skin from four volunteers was investigated for TGF-beta 1 and beta 2 immunostaining with antibodies that detect preferentially either cell- or matrix-associated forms of these peptides. Marked cell-associated TGF-beta 1 was found in the dermis, particularly around blood vessels and ducts; cellular TGF-beta 2 immunostaining was less prominent, and was predominantly around blood vessels. Neither TGF-beta 1 nor -beta 2 could be detected in the epidermis or epithelial structures, and the dermal matrix contained minimally detectable amounts of the two isoforms. In all cases, dermal matrix and cells contained greater amounts of TGF-beta 1 than TGF-beta 2. Previous studies have shown that both TGF-beta 1 and -beta 2 can induce dramatic increases in extracellular matrix, and both peptides have been implicated in the pathogenesis of fibrosis. We therefore investigated TGF-beta 1 and -beta 2 immunostaining in involved forearm skin of four patients with systemic sclerosis. Compared to normal skin, fibrotic specimens showed increased amounts of matrix and epidermal TGF-beta 1, but not TGF-beta 2. We conclude that TGF-beta 1 and -beta 2 expression in human skin is differentially regulated, and that their distribution is varied and complex.

Differences in the biological activities of transforming growth factor-beta and platelet-derived growth factor in vivo.

Transforming growth factor-beta 1 (TGF-beta 1 and recombinant platelet-derived growth factor-BB (rPDGF-BB) promoted an extensive, dose-dependent development of fibrous connective tissue when continuously delivered for 8 days by mini-osmotic pumps implanted subcutaneously in adult guinea pigs. Biochemical analyses demonstrated that TGF-beta 1 and rPDGF-BB stimulated dose-dependent increases in the dry weight, and protein, DNA, collagen, and glycosaminoglycan (GAG) contents of the fibrous connective tissue capsule that enveloped the pumps. The GAG/DNA mass ratio was markedly elevated by TGF-beta 1, but the collagen/DNA, protein/DNA, and collagen/protein ratios were not significantly increased. In contrast, rPDGF-BB generally decreased these mass ratios. Histological analyses suggested that this was due to the fact that rPDGF-BB induced a very cellular response with a marked influx of neutrophils and fibroblasts. TGF-beta 1 induced significantly less cellular response, which consisted primarily fibroblasts and macrophages. These results indicated that rPDGF-BB and TGF-beta 1 induced connective tissue deposition in vivo in a dose-dependent fashion, although the cellular nature of the responses as well as the structural composition of the extracellular matrices were clearly distinguishable between the two growth factors.

A novel polyclonal antibody (CL-B1/29) for immunolocalization of transforming growth factor-beta 2 (TGF-beta 2) in adult mouse.

A polyclonal antibody (CL-B1/29) raised against a synthetic peptide with an amino acid sequence identical to the first 29 N-terminal residues of bovine bone-derived transforming growth factor-beta 2 (TGF-beta 2) was characterized and used for immunolocalization of TGF-beta 2 in adult mice. Reduced staining of immunoblots and tissue after absorption of the antiserum with the immunizing peptide or with TGF-beta 2 but not with purified TGF-beta 1 demonstrated that the reagent is specific for TGF-beta 2, with little or no crossreactivity with TGF-beta 1. The immunolocalization of TGF-beta 2 was investigated in formalin-fixed, paraffin-embedded cultured cells and murine tissue. Specimens pre-digested with testicular hyaluronidase demonstrated immunostaining predominantly of extracellular connective tissue matrix, whereas specimens pre-digested with pronase E demonstrated primarily cytoplasmic staining. Immunoreactivity was widely distributed in connective tissue, muscle, adsorptive and secretory epithelia, especially of endocrine tissue, and neural tissue of adult mice.

Inhibition of connective tissue formation in dermal wounds covered with synthetic, moisture vapor-permeable dressings and its reversal by transforming growth factor-beta.

An investigation of synthetic, adherent, moisture vapor-permeable dressings (SAM) on dermal wounds healing by secondary intent has yielded the novel observation that SAM dressings severely inhibited the deposition of granulation tissue and subsequent collagenous tissue when compared with air-exposed wounds in mouse and guinea pig systems. Repair tissue was quantitated histomorphometrically in full-thickness wounds covered with SAM or left air-exposed for periods up to 3 weeks. Early in healing, mouse wounds left open to the atmosphere formed a scab which overlay a large volume of granulation tissue derived from two sources, one lateral, and the other deep and centrally located. In contrast, SAM-covered wounds contained only a small amount of granulation tissue which was derived solely from lateral sources. Granulation tissue was replaced by fibrous connective tissue over time, and this was always less in SAM-covered wounds. Deposition of large amounts of connective tissue in air-exposed wounds was associated with significant polymorphonuclear and mononuclear cell infiltrates, while the lack of granulation tissue formation in SAM-covered sites was associated with reduced inflammation. Dressing-induced inhibition of connective tissue could be partially reversed by treatment with transforming growth factor-beta form 1 or 2. Deposition of granulation tissue in large lenticular wounds in guinea pig skin, but not in 6-mm punch wounds, was also inhibited when the wounds were covered with SAM, and the morphology of air-exposed and SAM-covered wounds was similar to that in mice. SAM-covered wounds in mice and guinea pigs may be useful as models of chronic non-healing wounds.

Light and electron microscope analysis of lectin binding to adult rat liver in situ.

A comprehensive mapping of lectin receptors on adult rat liver in situ was performed at light and ultrastructural levels by using 12 biotin-labeled lectins and an avidin-biotin-peroxidase complex. In addition, concanavalin A conjugated directly to peroxidase was utilized to study intracellular membrane glycoconjugates. To achieve optimal preservation of these membrane sugar moieties, several fixatives and fixation procedures were evaluated. A periodate-lysin-paraformaldehyde combination provided the best compromise between preservation of ultrastructural details and lectin-binding reactivity. Hepatocyte cell surfaces reacted intensely with concanavalin A, Lens culinaris agglutinin, and Pisum sativum agglutinin (all specific for alpha-D-mannosyl and alpha-D-glucosyl groups) as well as Ricinus communis agglutinin type I (specific for alpha or beta-D-galactose) and wheat germ agglutinin (specific for neuraminic acid and beta-NAc-glucosaminyl groups). In addition, R. communis agglutinin and wheat germ agglutinin exhibited an extremely strong reactivity for bile canaliculi which surpassed the binding of concanavalin A, L. culinaris agglutinin, and P. sativum to these structures. Phaseolus vulgaris agglutinin (specific for beta-D-galactose-glucosyl-NAc and D-mannosyl groups), which exhibited a moderate binding to hepatocyte plasma membranes, reacted more strongly with the endothelium of sinusoids and portal vessels. Although all six of these lectins plus Bandeiraea simplicifolia stained Kupffer cells, B. simplicifolia lectin (an alpha-D-galactosyl marker) was unique in showing a strong reactivity for only this cell type. The avidin-biotin-peroxidase procedure is a sensitive method for detection of sugar moieties on cell surfaces of rat liver at both light and electron microscopic levels. In this study, the procedure was used to localize differential binding of lectins to several anatomical structures of the organ, and furthermore, we were able to map preferential localizations of carbohydrate residues in the glycocalyx of the rat hepatocyte in situ.

Congenic strains of RCS rats with inherited retinal dystrophy.

Two congenic strains of RCS rats, RCS-p/+ and RCS-c, have been developed that differ from the parental strain at genetic loci affecting pigmentation. Inbred RCS rats are pink-eyed, while RCS-p/+ rats produce segregating litters of pink-eyed (p/p) and black-eyed (p/+) offspring, and RCS-c rats are albinos. All the strains are homozygous for the mutant form of the retinal dystrophy gene. The black eye pigment in RCS-p/+ rats slows the progression of the retinal degeneration by about 10 days in the posterior retina and by about 30-35 days in the peripheral retina in the superior half of the eye. No slowing of the disease occurs in the inferior half of the eye along the vertical meridian. All the strains are similar in body weight and litter size, and show a low incidence of cataract and microphthalmia.