Stimulation of granulation tissue formation by platelet-derived growth factor in normal and diabetic rats.

Subcutaneous implantation of Hunt-Schilling wound chambers in rats induces a wound repair response causing the chamber first to fill with fluid and subsequently with connective tissue. The presence of a type I collagen gel encouraged a more rapid dispersion of cells throughout the chamber but had no effect on the rate of new collagen deposition. Addition of platelet-derived growth factor (PDGF; 50 ng/chamber) to the collagen-filled chambers caused an earlier influx of connective tissue cells, a marked increase in DNA synthesis, and a greater collagen deposition in the chamber during the first 2 wk after implantation. After 3 wk, however, the levels of collagen were similar in PDGF-supplemented and control chambers. Diabetic animals exhibited a decreased rate of repair which was restored to normal by addition of PDGF to the wound chamber. Combinations of PDGF and insulin caused an even more rapid increase in collagen deposition. These results suggest that the levels of various growth factors, particularly PDGF, may be limiting at wound sites and that supplementation of wounds with these factors can accelerate the rate of new tissue formation.

PINP: a serum biomarker of bone formation in the rat.

Serum PINP has emerged as a reliable marker of bone turnover in humans and is routinely used to monitor bone formation. However, the effects of PTH (1-34) on bone turnover have not been evaluated following short-term treatment. We present data demonstrating that PINP is an early serum biomarker in the rat for assessing bone anabolic activity in response to treatment with PTH (1-38). Rat serum PINP levels were found to increase following as few as 6 days of treatment with PTH (1-38) and these increases paralleled expression of genes associated with bone formation, as well as, later increases in BMD. Additionally, PINP levels were unaffected by treatment with an antiresorptive bisphosphonate. PINP may be used to detect PTH-induced early bone formation in the rat and may be more generally applicable for preclinical testing of potential bone anabolic drugs.

Doxorubicin-induced impairment of wound healing in rats.

The mortality rate induced by 3 doses of iv doxorubicin was evaluated in F344 rats, and a dose of 8 mg doxorubicin/kg body weight was the maximum dose tolerated with an acceptable mortality rate. Rats treated with 8 mg doxorubicin/kg prior to or on the day of wounding demonstrated decreased wound breaking strength in incisional wounds at all intervals after wounding. Decreased amounts of collagen and DNA and less cellularity were noted in wound chambers from rats treated in the same manner. In both the incisional wound and wound chamber models, rats treated with doxorubicin 7 days after wounding showed a less dramatic healing impairment. No difference in collagen types was noted between chambers from the doxorubicin-treated and untreated rats. Doxorubicin also produced a significant reduction in platelet and white blood cell counts 1 week after it was administered. The data indicate that doxorubicin impedes healing by decreasing wound cellularity and collagen synthesis.

Osteonectin/SPARC is a product of articular chondrocytes/cartilage and is regulated by cytokines and growth factors.

Rabbit articular chondrocytes maintained in monolayer, synthesized and secreted a 46 kDa protein into the culture medium. N-terminal sequence analysis and immunoprecipitation of the radiolabeled material revealed this protein to be osteonectin (ON)/SPARC, a protein previously shown to be present in bone. When chondrocytes were exposed to interleukin-1, a cytokine with matrix degradative properties, ON synthesis and secretion was greatly inhibited. However, this was specific to IL-1 since two other pro-inflammatory cytokines (tumor-necrosis factor-alpha and interleukin-6) with properties similar to IL-1, failed to cause any discernible effect on ON synthesis. Several growth factors (TGF-beta, PDGF, and IGF-1), that have been shown to stimulate other cartilage matrix macromolecular synthesis, also stimulated ON synthesis and were also able to reverse the inhibitory effect of IL-1 on ON synthesis. These observations were also demonstrated in explant cultures of cartilage. Our studies suggest that ON is a biosynthetic product of articular cartilage and could play a role in cartilage structure and/or function.

Comparison of recombinant human PTH(1-34) (LY333334) with a C-terminally substituted analog of human PTH-related protein(1-34) (RS-66271): In vitro activity and in vivo pharmacological effects in rats.

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are believed to exert their biological actions through binding and activation of a common cell surface receptor. Recently, an analog of PTHrP (RS-66271), was described that demonstrated reduced binding affinity for the PTH/PTHrP receptor compared with bovine PTH(1-34) but retained equal biological activity. The present study investigated the receptor binding affinities of synthetic RS-66271 and recombinant human PTH(1-34) (LY333334) and compared their in vitro and in vivo pharmacological effects. RS-66271 had one hundredth the activity of PTH(1-34) in competing for the binding of [125I] [Nle8,18, Tyr34]human PTH(1-34) to the human PTH/PTHrP receptor stably expressed in a human kidney cell line. Despite this reduced binding affinity, RS-66271 had equivalent activity in increasing both cAMP production in osteoblast-like cells and bone resorption in neonatal mouse calvariae. However, RS-66271 was 7. 6-fold less active in stimulating inositol phosphate production. For in vivo studies, young, male Fisher rats received a daily subcutaneous dose of either 10 or 40 microg/kg of peptide for 1, 2, or 4 weeks. Volumetric bone mineral density and total bone mineral content of the proximal tibia were determined by peripheral quantitative computerized tomography. Trabecular and cortical bone of the distal femur were analyzed for calcium and dry weight. Lumbar vertebrae (L4-L6) were analyzed by histomorphometry. Trabecular and cortical bone mass showed a dose- and time-dependent increase in the treated animals compared with the controls. These increases were evident as early as 1 week after initiation of dosing. There were no consistent significant differences in the comparative effects of PTH(1-34) and RS-66271 on the measured bone parameters. In conclusion, despite the reduced binding affinity of RS-66271 for the PTH/PTHrP receptor compared with human PTH(1-34), both peptides displayed similar in vitro and in vivo pharmacological effects.

Differential regulation of metalloprotease steady-state mRNA levels by IL-1 and FGF in rabbit articular chondrocytes.

The expression of collagenase and stromelysin is believed to be coordinately regulated. In this report however, we provide evidence that suggests subtle differences may exist in the early events of the induction of these enzymes. Rabbit articular chondrocytes treated with interleukin-1, either alone or in combination with fibroblast growth factor, accumulated steady-state mRNA levels for both the enzymes, with the latter treatment more effective in inducing greater levels and within a shorter time. Further, the induction of the enzymes by either protocol was blocked by cycloheximide co-treatment. Cycloheximide added 1 h post-stimulation with interleukin-1 + fibroblast growth factor failed to block stromelysin mRNA expression, but was able to block collagenase steady-state mRNA levels. Transforming growth factor-beta, another inhibitor of metallprotease induction, showed no such differential activity. The results suggest that collagenase and stromelysin may have subtle variations in their induction pathways. Our studies further show that the enzyme induction by interleukin-1 alone or in combination with fibroblast growth factor occurs through different, but related mechanisms.

Proliferating cells in the primary spongiosa express osteoblastic phenotype in vitro.

We have shown that intermittent parathyroid hormone (PTH) treatment targets proliferating cells in the primary spongiosa of trabecular bone of young rats, resulting in an increased number of osteoblasts. To further characterize these proliferating osteoprogenitor cells, bromodeoxyuridine (BrdUrd) incorporated in vivo, was used as a marker to identify and isolate cells for in vitro studies. Proliferating cells were labeled in vivo in young rats with BrdUrd and 24 h later were isolated by trypsinization of sections of the primary spongiosa of the distal femur metaphysis. Within 12 h of isolation, BrdUrd+ cells formed distinct foci containing 20-500 cells with fibroblast morphology. Stimulation of proliferation as determined by [3H]-thymidine incorporation was observed for these cells in response to fetal bovine serum, platelet derived growth factor, and transforming growth factor beta-1. Neither insulin-like growth factor-1 (IGF-1) nor insulin stimulated proliferation PTH (1-34) and dexamethasone inhibited proliferation. The effects of PTH and dexamethasone were additive. Cells expressed the osteoblast phenotype as evidenced by synthesis of type I collagen, expression of high alkaline phosphatase activity, and production of increased intracellular cAMP in response to PTH (1-34). Confluent cell aggregates spontaneously formed mineralized nodules within 4-7 days, in the absence of inducers. These observations suggest that the primary spongiosa cells recapitulates the differentiation process in vitro in an accelerated fashion and may serve as a useful model to study osteoblast differentiation.

Chemotaxis of rat retinal glia to growth factors found in repairing wounds.

Retinal glia migrate to sites in the vitreous body and on the inner and outer surfaces of the retina during a wide variety of pathological processes. Using in vitro cultures of rat retinal glia, we have evaluated an extensive list of homogeneous, well-characterized growth factors as chemoattractants for these cells. Humoral growth factors, fibroblast growth factors, extracellular matrix proteins and immune system cytokines all were without activity except for the platelet-derived growth factor (PDGF). Extracts of bovine ocular tissues including vitreous body and retina yielded significant activity.

Primary culture of human retinal glia.

Glial cells of the human retina participate in various pathologic processes characterized by cell migration, proliferation, and extracellular matrix production. To study these events in vitro, a procedure was developed to obtain primary cultures of human retinal glial cells. The cultures resulting from the processing of 130 globes contained cells with variable morphology including bipolar and multipolar or stellate cells. Most cells in the primary culture were labeled with antisera to the glial fibrillary acidic protein. The cultures were also examined with antibodies directed against factor VIII-related antigen and muscle-specific actin to determine the presence of endothelial cells and pericytes. A variable contamination of cells staining for the latter was found in these cultures (usually less than 10%). Together, these data indicated that the primary cultures arose principally from glial cells of the human retina but did not precisely identify the cell of origin.

Modulation of PDGF mediated osteoblast chemotaxis by leukemia inhibitory factor (LIF).

Cell migration is a key event in tissue repair and remodeling. PDGF, a growth factor for multiple target cells, has been shown to be a potent chemoattractant for a variety of mesenchymal cells. However, it is likely that PDGF-mediated cell migration will be influenced by other cytokines that can be produced during physiological and pathological conditions. Leukemia inhibitory factor (LIF), a cytokine that is produced by a variety of cells including osteoblasts, may promote bone formation, but the mechanism is not known. Since osteoblasts are responsible for laying down new matrix during skeletal remodeling, in this report we have examined whether PDGF or LIF influences the migration of osteoblasts. Among several cytokines and growth factors tested, only PDGF was able to elicit a major chemotactic (directed migration) and a minor chemokinetic (random-migration) response in osteoblasts. LIF alone was not active in either chemotaxis or chemokinesis but when included with PDGF it caused a reduction in chemokinesis. Further, pretreatment of osteoblasts with LIF caused an increase in PDGF-driven chemotaxis. Finally, osteoblasts exposed briefly to LIF synthesized a higher level of non-collagenous proteins upon further treatment with PDGF. These observations are consistent with a role for LIF in promoting bone formation, both by influencing directional migration of osteoblasts and in laying down new matrix.

Synthesis of type IX collagen: effect of beta-xylosides.

Type IX collagen contains a chondroitin sulfate side chain and therefore may be considered as a proteoglycan. We investigated the effect of beta-xylosides on type IX collagen synthesis. Treatment of chondrocytes with beta-xylosides results in the loss of synthesis of large and small molecular weight proteoglycans, but the synthesis of type IX collagen was unaffected. It is likely that the mechanism of addition of sugar residues to type IX collagen is distinct from that of other cartilage proteoglycans.

Transforming growth factor-beta is a potent inhibitor of IL-1 induced protease activity and cartilage proteoglycan degradation.

Treatment of chondrocytes in culture with interleukin-1 results in the production of neutral proteases that cause the degradation of the large aggregating proteoglycan. TGF-beta is a pleiotropic growth factor that has been shown to induce differentiation of cartilage and, in some cases, was able to inhibit the IL-1-dependent processes. In this report, we examined whether TGF-beta could block the IL-1 induced catabolic effects on chondrocytes. After treatment with IL-1 beta (30 ng/ml), rabbit articular chondrocytes produced approximately 2 units of neutral protease activity. Under identical conditions, TGF-beta 1 alone did not induce any protease activity. However, a combination of IL-1 and TGF-beta resulted in a dramatic reduction in the level of protease activity. The inhibitory effect of TGF-beta was also observed at the level of proteoglycan incorporation into the extracellular matrix. The IL-1 treated chondrocytes failed to incorporate proteoglycans into their extracellular matrix. However, addition of TGF-beta in the presence of IL-1 resulted in partial reversal towards a normal extracellular matrix. These studies indicate that TGF-beta can block and at least partially inhibit the catabolic effects of IL-1 on chondrocytes.

Induction of interleukin-1 receptors on chondrocytes by fibroblast growth factor: a possible mechanism for modulation of interleukin-1 activity.

Interleukin-1 is a polypeptide factor with profound effects on several cell types, such as chondrocytes, fibroblasts, and T-cells. The ability of interleukin-1 to induce the synthesis of matrix-degradative enzymes, as well as prostaglandin E2, suggests a pivotal role for this mediator in chronic inflammation. Previous studies have shown that the effect of human monocyte interleukin-1 on the synthesis of collagenase and neutral proteases by chondrocytes was enhanced by basic fibroblast growth factor. Using recombinant human interleukin-1B, we have examined whether the potentiation of interleukin-1 effects by fibroblast growth factor is related to changes in the number or affinity of interleukin-1 receptors. Our studies confirm that rabbit articular chondrocytes in culture contain a single class of high-affinity receptors for interleukin-1 with a Ka of 0.9-1.1 x 10(-13) M-1. While the untreated chondrocytes contain approximately 1,620 receptors per cell, fibroblast growth factor-treated cells exhibit a higher number of receptors (approximately 2,960 per cell) with no apparent change in the affinity. The increase in receptor number can be abolished by inhibitors of lysosomal function, indicating a requirement for intracellular processing of the fibroblast growth factor. Our results suggest that the potentiation of interleukin-1 catabolic effects by fibroblast growth factor may be related to its ability to induce additional interleukin-1 receptors on the chondrocyte cell surface.