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Platelet-derived growth factor (PDGF) has been implicated in the processes regulating gliogenesis in the CNS. Conflicting in vivo data in rodents have variously implicated either glia or neurons as being the primary source of PDGF. We have used in situ hybridization and immunocytochemical analysis to study the in vivo expression and cellular localization of PDGF-A, sis/PDGF-B, together with the two PDGF receptors alpha and beta, in developing human forebrain. In this study we demonstrate the strong expression of mRNA and protein of both PDGF chains, A and B, and their receptors, alpha and beta, in human embryonic glial cells. The neurons, in contrast to glial cells, expressed lower levels of PDGF and PDGF-receptor mRNAs and protein. Identification of the cell types expressing the PDGF and PDGF-receptor mRNAs was achieved by counterstaining with antibodies specific for glial cells (GFAP) and neurons (NF). The predominant glial-specific expression of both PDGF-A and PDGF-B, together with the coexpression of their receptors alpha and beta, suggests an important role for the PDGF isoforms in the development of human embryonic glial cells and neurons in vivo.

The repair of nerve gap injuries with tubular nerve guides has been used extensively as an in vivo test model in identifying substances which may enhance nerve regeneration. The model has also been used clinical nerve repair. The objective of this study was to compare three different gel matrix-forming materials as potential vehicles for growth factors in this system. The vehicles included a laminin containing extracellular matrix preparation (Biomatrix), collagen, and a 2% methylcellulose gel. The growth factor test substance consisted of a combination of platelet-derived growth factor BB (PDGF-BB) and insulin-like growth factor I (IGF-I). An 8-mm gap in rat sciatic nerve was repaired with a silicone tube containing each of the vehicles alone or with a combination of each vehicle plus PDGF-BB and IGF-I. At 4 weeks after injury, the application of the growth factor combination significantly stimulated axonal regeneration when applied in methylcellulose or collagen, but not in Biomatrix. A similar trend was present between the vehicle control groups. By 8 weeks after injury, nerves repaired with methylcellulose as a vehicle had significantly greater conduction velocity than either collagen or Biomatrix. It was concluded that a 2% methylcellulose gel was the best of the three matrices tested, both in its effects on nerve regeneration and flexibility of formulation.

There has been considerable interest in the potential role of growth factors in the initiation and development of cutaneous malignant melanoma (CMM). Platelet-derived growth factor (PDGF) has been shown to be secreted by melanoma cell lines and by metastatic melanoma in vivo. PDGF also has been reported to stimulate the development of tumour stroma and new blood vessels. We studied the expression of PDGF and its receptors by both immunohistochemistry (IHC) and in situ hybridization (ISH) in primary and metastatic melanoma and in normal skin specimens. Cryostat sections were incubated with 35S-labelled riboprobes and antibodies for PDGF-AA, PDGF-alpha receptor, PDGF-BB and PDGF-beta receptor. Both primary and metastatic melanoma exhibited significant expression of PDGF-AA, PDGF-BB and PDGF-alpha receptor by both IHC and ISH, compared with only background expression in normal skin. We did not observe expression of PDGF-beta receptor in melanoma. Our results suggest that PDGF may function as an autocrine growth factor, as well as an angiogenesis factor, in CMM tumour development. This expression of the PDGF-alpha receptor rather than the beta receptor may be unique among solid tumours.

The antiproliferative flavonoid, quercetin, is limited in its pharmacological utility by its low water solubility. In this paper, we describe the synthesis of two quercetin analogues prepared by linking the hydroxyl group at the 3- or 5-position of the flavonoid to the 1-hydroxyl group of myo-inositol-2-phosphate via a succinate diester linkage. The resulting conjugates were found to have dramatically enhanced water solubility relative to quercetin; the 5-linked quercetin analogue 2 had a water solubility of > 300 mg/mL at 20 degrees C. Comparison of the in vitro cytotoxicity and antiproliferative activity of conjugate 2 with those of quercetin toward cultured human colon adenocarcinoma (SW480) and human glioblastoma (U87MG) cells indicated that this modification of quercetin does not significantly diminish its activity in these assays.

Platelet-derived growth factor (PDGF), an osteoblast mitogen, has been demonstrated to accelerate fracture healing and periodontal bone repair when applied locally in vivo. To explore whether PDGF could stimulate bone formation in intact bone, we administered it systemically to rats rendered acutely estrogen-deficient. Because PDGF may stimulate bone resorption in vitro, PDGF was administered with and without an antiresorptive agent (alendronate). All treatments were given by intravenous injection 3 times a week for 6 weeks. Spinal bone mineral density (BMD) decreased by 5% in the vehicle-treated ovariectomized (OVX) rats by the end of the study as determined by DXA. Treatment with PDGF prevented this bone loss and significantly (p < 0.05) increased the bone density in the spine (9%) and whole skeleton (5.8%). Combined treatment with PDGF and alendronate resulted in a greater increase at the spine (18%) and whole skeleton (12.8%) than either agent alone. Histomorphometric analysis demonstrated that treatment with PDGF increased the osteoblast number and osteoblast perimeter without consistent changes in osteoclast estimates. Biomechanical testing demonstrated that PDGF administration increased the vertebral body compressive strength and femoral shaft torsional stiffness and resulted in a trend for enhanced femoral head shearing strength. Coadministration of alendronate further increased these indices of bone strength. PDGF administration also caused premature closure of the growth plate, decreased body fat, and resulted in extraskeletal collagen deposition. We therefore demonstrate, for the first time, that systemic administration of PDGF can increase bone density and strength throughout the skeleton.

Abasic (AP) sites in DNA are cytotoxic and mutagenic and their repair is initiated by AP endonucleases. The major AP endonuclease of mammalian cells is encoded by the APE gene. Ape protein has also been proposed to modulate the activity of some transcription factors independently of its AP endonuclease activity. We investigated whether APE expression is coordinated with cell division, which could diminish mutagenesis. The level of APE mRNA was followed during wound healing in porcine epidermis, in which surgical wounding prompts rapid cell proliferation followed by a differentiation program to regenerate normal skin. In situ hybridization with a probe from human APE cDNA revealed strongly decreased expression in rapidly proliferating migrating cells during the first 1-3 days following wounding, succeeded by sharply increased APE expression that exceeded the pre-wounding levels by days 9-17. These changes were not observed in the surrounding undamaged tissue. In contrast to the foregoing in vivo results, APE expression in cultured primary human fibroblasts (IMR90) or myeloid leukemia cells (K562) was not coordinated with cell division. This biphasic APE expression during wound healing could relate to transcription factor regulation or it could allow unhindered DNA synthesis or prepare the developing epidermis to handle DNA damage. However, if transient under-expression of APE-encoded repair enzyme does occur, it might render regenerating skin especially vulnerable to mutagenesis during the cell proliferation phase.

Platelet-derived growth factor and insulin-like growth factor-I have been shown to interact synergistically to enhance repair of skin wounds in normal healing swine. Platelet-derived growth factor alone has shown promise in treating human chronic ulcers. The objective of this study was to compare the wound healing effects of platelet-derived growth factor-BB alone with those of a combination of platelet-derived growth factor-BB and insulin-like growth factor-I in an improved model with the use of "older" animals with diabetes. Older diabetic (db/db) mice (>15 weeks of age) have less elevated insulin levels compared with young db/db mice. The serum insulin levels in the older animals is 1.0 to 2.5 times that of the nondiabetic animals, a similar increase to that which occurs in human patients with type II diabetes. Healing was evaluated in two studies involving a total of 104 animals. Treatment groups included the following: 4.0 microg/cm(2) of platelet-derived growth factor-BB, 40.0 microg/cm(2) of platelet-derived growth factor-BB, 4.0 microg/cm(2) of both platelet-derived growth factor-BB and insulin-like growth factor-I or vehicle. All growth factors were applied topically in a methylcellulose vehicle to full-thickness wounds every other day for 24 days. Efficacy end points were median and mean time to complete healing and rate of wound closure. The median time to complete healing for animals receiving the platelet-derived growth factor-BB/insulin-like growth factor-I combination was 38% and 33% faster (p < 0.001) than animals receiving 4.0 microg/cm(2) and 40.0 microg/cm(2) of platelet-derived growth factor-BB, respectively. The mean time to complete healing for platelet-derived growth factor/insulin-like growth factor-I treated animals was 31% and 29% faster (p < 0.001) than 4.0 microg/cm(2) and 40.0 microg/cm(2) platelet-derived growth factor-BB treated animals, respectively. Wounds treated with 4.0 microg/cm(2) platelet-derived growth factor-BB/insulin-like growth factor-I healed, on average, in 22 days compared with 31 days for 40.0 microg/cm(2) platelet-derived growth factor-BB alone and 38 days for vehicle. Also, platelet-derived growth factor-BB/insulin-like growth factor-I significantly improved the rate of wound closure throughout the duration of the studies compared with either dose of platelet-derived growth factor-BB alone (p < 0.005) or vehicle (p < 0.001). In conclusion, the data show that the combination of platelet-derived growth factor-BB and insulin-like growth factor-I is more effective than platelet-derived growth factor-BB alone at the doses tested or vehicle treatment in stimulating cutaneous wound healing in older, diabetic mice.

The present studies investigated the in vivo expression of the p53 suppressor gene and protein product in response to acute cutaneous injury in swine, along with the parallel expression of the c-sis/PDGF-B mitogen and its receptor beta (PDGF-R beta). p53 expression was shown to be suppressed during the period of active cellular proliferation in the injured tissue and to reemerge during the stages of healing. In contrast, c-sis/PDGF-B and PDGF-R beta were expressed during the early phase of active cellular proliferation and they were suppressed upon healing. This inverse relationship between mitogenic growth factors and p53 suggests the presence of well-controlled physiologic mechanisms that regulate in vivo the processes of normal tissue repair in response to injury. At the stages of tissue regeneration, these mechanisms include both the expression of growth factors that promote cell proliferation and the suppression of p53 that downregulates proliferation. At the stages of healing, the expression of the mitogenic growth factors is suppressed and that of p53 reemerges, reaching its peak at the time of complete epithelialization and healing of the injured tissue. These studies are the first to link the response of p53 protein to physiologic processes of tissue regeneration in vivo.

Gingival inflammation is initiated by bacterial colonization on the tooth surface. It is characterized by infiltration of mononuclear cells, a common feature of many forms of chronic inflammation. Monocyte chemoattractant protein 1 (MCP-1) is the predominant monocyte chemoattractant secreted by a variety of different cells in vitro. For this report, we examined MCP-1 expression in bacterially induced gingival inflammation by immunohistochemistry and in situ hybridization. The cell types expressing MCP-1 are identified as vascular endothelial cells and monocytes/macrophages. Correlation analysis shows that the number of cells expressing MCP-1 is related to the degree of inflammation. Our finding that MCP-1 is expressed in inflamed gingival tissue suggests that MCP-1 plays an important role in the recruitment of monocytes and amplification of inflammatory signals in bacterially induced inflammation.

We report that acute injury induces the expression of selective growth factor and growth factor receptors in the epithelial cells of the wounded tissue. In situ hybridization analysis of skin biopsy specimens obtained after cutaneous injury in swine demonstrated the induction of the expression of transforming growth factor-alpha, its receptor, epidermal growth factor-R, acidic fibroblast growth factor, and basic fibroblast growth factor messenger RNAs in the skin epithelial cells of the wounded tissue. There was no significant expression in the epithelial cells of control, uninjured tissues. The expression levels were maximal during the period of active tissue repair (1 to 5 days after injury) and were totally suppressed upon the healing of the wounded tissues. In contrast, insulinlike growth factor-I, (IGF-I), IGF-I receptor, and IGF-II receptor messenger RNAs were expressed in the epithelial cells of both the control, uninjured tissues and in tissue specimens obtained after injury. There was no significant expression of IGF-II messenger RNA in the epithelial cells before or after injury. It seems that injury induces the coordinated expression of selective growth factor and growth factor receptor genes whose products contribute to the regulation of the complex processes involved in tissue repair and remodeling.

Meningiomas are common brain tumors that show a predilection for females and become more aggressive during pregnancy and menses. The existence of gender-specific hormone receptors in meningiomas has long been a matter of controversy; the recent cloning of androgen, estrogen, and progesterone receptors has facilitated their direct evaluation. The authors have demonstrated the expression of androgen and progesterone receptor messenger ribonucleic acid and protein product in nine primary human meningiomas by Northern blot analysis. Cellular localization was achieved by in situ hybridization analysis. Estrogen receptor expression was not detected. Normal adult meninges were shown to express very low levels of both androgen and progesterone receptors.

Atherosclerosis is marked by an overt inflammatory infiltrate, with enhanced recruitment of monocytes/macrophages observed in both human and experimental atherosclerosis. We previously determined that monocyte chemoattractant protein 1 (MCP-1) accounts for virtually all of the chemotactic activity produced by vascular (aortic) smooth muscle cells in culture. We now report that arteries from a primate model of atherosclerosis with dietary-induced hypercholesterolemia exhibit increased levels of MCP-1 mRNA expression in vivo, whereas their normal counterparts demonstrate minimal MCP-1 expression. Furthermore, immunohistochemistry and in situ hybridization clearly indicate that the expression of MCP-1 protein and mRNA is in the smooth muscle cells of the medial layer of the artery and in monocyte-like and smooth muscle-like cells found in the overlying intimal lesion. These studies indicate that one of the responses to dietary hypercholesterolemia is the expression of MCP-1 by vascular smooth muscle cells. This expression, when augmented with other cellular and molecular factors, could significantly contribute to the recruitment of monocytes/macrophages to the vessel wall.

Macrophages are thought to play an important role in the pathologic changes associated with idiopathic pulmonary fibrosis (IPF). The mechanisms for increased monocyte/macrophage recruitment in IPF are unknown. Monocyte chemoattractant protein 1 (MCP-1) is the predominant monocyte chemoattractant secreted by a variety of different cell types in culture. We examined the expression of MCP-1 mRNA and its protein product in vivo in IPF and non-IPF lung specimens by in situ hybridization and immunocytochemistry. The cell types expressing MCP-1 in vivo were identified by immunostaining with specific antibodies. We demonstrated the expression of MCP-1 mRNA in pulmonary epithelial cells, in monocytes/macrophages, and in vascular endothelial and smooth muscle cells. Lung epithelial cells in patients with IPF strongly expressed MCP-1 mRNA and its protein product. In contrast, epithelial cells in non-IPF specimens did not express MCP-1 mRNA. Macrophages and vascular endothelial and smooth muscle cells were shown to express MCP-1 in both IPF and non-IPF lung specimens. These findings provide a basis for the understanding of the in vivo physiologic processes that mediate monocyte/macrophage recruitment and infiltration in the lung interstitium and the pathologic state contributing to an increased alveolar monocyte/macrophage population and inflammation in IPF.

In situ hybridization and immunocytochemistry have been applied to investigate the expression of c-sis/platelet-derived growth factor (PDGF)-B, insulin-like growth factor (IGF)-I, and transforming growth factor alpha mRNAs and their respective receptor mRNAs in three primary human gastric carcinomas and in their adjacent nonmalignant mucosas. Expression of c-sis/PDGF-B mRNA and PDGF-receptor beta mRNA was seen in the tumor cells of the three gastric cancer specimens but not in their adjacent nonmalignant mucosa. The mRNA expression was accompanied by the expression of their respective protein products. IGF-I, IGF-I receptor, and epidermal growth factor receptor mRNAs were seen in both the tumor cells of the gastric cancer specimens and in nonmalignant mucosa. Transforming growth factor alpha mRNA was expressed in gastric tumor cells but not in nonmalignant mucosa. The coexpression of a potent "competence" growth factor, PDGF, and "progression" growth factors, IGF-I and transforming growth factor alpha, in the tumor cells of gastric carcinomas may contribute to their growth and maintenance.

Glioblastomas are malignant brain tumors that are attended by an immunosuppressed state. The authors have studied the expression of transforming growth factor-beta 2, which is known to have potent immunosuppressive and angiogenic properties. Transforming growth factor-beta 2 messenger ribonucleic acid and its protein product are both found to be greatly overexpressed in these tumors and are absent from normal brain tissue. The overexpression of this growth factor may contribute to the escape of neoplastic astrocytes from immune surveillance and, furthermore, to the immunosuppressed state that is characteristic of many of these patients.

Lung cancer represents one of the major human carcinomas with the highest degree of mortality. Epidemiologic studies have linked this disease to "chronic injury," largely induced by cigarette smoking. In the present studies, we demonstrate the in vivo expression of platelet-derived growth factor (PDGF) and PDGF receptor (PDGF-R) beta mRNAs and their respective protein products in malignant epithelial cells of primary human lung carcinomas. In contrast, nonmalignant epithelial cells in control, normal lung tissue specimen did not express PDGF and PDGF-R mRNAs and did not produce their respective protein products. Epithelial cells in lung specimen from patients with idiopathic pulmonary fibrosis expressed only PDGF mRNA but not PDGF-R beta mRNA. These findings of the inappropriate coexpression of a potent mitogen, PDGF, and its receptor in lung cancer epithelial cells suggest the presence of a powerful in vivo mechanism contributing to the self-stimulation and unregulated growth of lung cancer tumor cells.

These studies demonstrate the expression of IGF-1, IGF-11, and their respective receptor mRNAs in primary human astrocytomas and meningiomas. In situ hybridization and immunocytochemistry have localized a strong expression of both IGF-1 and IGF-11 mRNAs and of their protein products in the tumor cells of astrocytomas and meningiomas. The expression of IGF-1 and IGF-11 mRNAs in the tumor cells was accompanied by the co-expression of their respective type-1 and type-11 IGF receptor mRNAs. Control, non-malignant human brain expressed IGF-1 mRNA and IGF-1 and IGF-11 receptor mRNAs. There was no significant expression of IGF-11 MRNA in the control brain specimens. Control pachymeninges (dura mater) expressed low levels of IGF-1 mRNA and IGF-1 receptor mRNA. There was no significant expression of IGF-11 and IGF-11 receptor mRNAs in pachymeninges. The co-expression of IGFs and their receptors in brain tumors may contribute in their development and maintenance. The strong inappropriate expression of IGF-11 mRNA and its protein product in the tumor cells of astrocytomas and meningiomas, but not in normal brain specimens, may serve as molecular markers for the early detection of these tumors.

Ten patients with aggressive fibromatosis of the extremities were prospectively followed for 2-6 years. Results of treatment methods were compared. Five patients underwent three-dimensional imaging with and without intravenous contrast, and the images were compared with the anatomic extent of the resected lesion. Pathologic specimens and control tissue were tested for the presence of estrogen and progesterone receptors and for expression of the c-sis oncogene and platelet-derived growth factor (PDGF), potent mitogens for fibrocytes. Wider surgical resection resulted in a lower recurrence rate, but current chemotherapeutic agents were not effective in eradicating the tumors. Intravenous contrast enhanced the lesions. Two thirds of the tumors tested had estrogen or progesterone receptors. All tumors tested had inappropriate expression of c-sis and PDGF. This inappropriate expression may be responsible for the underlying pathobiology and deregulation of control of growth in aggressive fibromatosis.

A common feature of human melanoma is infiltration by monocytes at early stages of tumorigenesis. This infiltration may be highly significant since macrophages have the capacity to alter the behavior of tumor cells. The authors previously demonstrated that the predominant monocyte chemoattractant produced by tumor cells in vitro was monocyte chemotactic protein-1 (MCP-1). The authors identify the expression of MCP-1 in pathologic specimens of both primary and metastatic human melanoma but not in normal skin. The finding that MCP-1 is produced by malignant melanoma suggests that specific genes are expressed in tumor cells that can induce the recruitment of monocytes in vivo.

hPDGF is the major growth factor of human blood serum. In vivo, it is apparently synthesized by megakaryocytes and is transported in blood stored in the alpha granules of platelets. hPDGF is a heterodimer of two homologous polypeptide chains (PDGF-1(A) and PDGF-2(B] linked together by disulphide bonds. The PDGF-1(A) chain is encoded by a gene localized in chromosome 7 and the PDGF-2(B) chain is encoded by the c-sis proto-oncogene localized in chromosome 22. The hPDGF heterodimer and its two isoforms, the PDGF-1(A) and PDGF-2(B) homodimers, are potent mitogens and chemoattractants for target cells such as diploid fibroblasts, osteoblasts, arterial smooth muscle cells and brain glial cells. The PDGF-1(A) homodimer binds only to its specific receptor alpha, and the hPDGF heterodimer and PDGF-2(B) homodimer bind to both receptors a and b. In addition to their mitogenic action, PDGF stimulates important cellular metabolic activities, including protein, lipid and prostaglandin synthesis. It appears to be an important factor in early development and in vivo appears to modulate tissue regeneration and remodelling during wound healing and osteogenesis. The inappropriate expression of PDGF genes and their mitogenic products has been linked to several proliferative disorders such as fibrosis, atherosclerosis and neoplasia.

Bone has the capacity for repair and regeneration. The repair process is thought to be locally regulated by growth factors. One of the growth factors that potentially plays a significant role in these processes is platelet-derived growth factor (PDGF). Two different PDGF genes have been identified, PDGF-A and PDGF-B, whose gene products give rise to biologically active dimers. We now report that PDGF-AA and PDGF-BB exhibit saturable binding to normal human osteoblastic cells. By Scatchard analysis we estimate that there are approximately 43,000 PDGF-AA binding sites per cell, with a dissociation constant (Kd) of 2.2 x 10(-10)M, and 55,000 high-affinity PDGF-BB binding sites per cell, with a Kd of 1.2 x 10(-10)M. The functional consequence of PDGF binding was also assessed. PDGF-AA and PDGF-BB both stimulated migration of normal human osteoblastic cells and stimulated thymidine incorporation. To gain insight into potential transmodulation of the PDGF response, we investigated the capacity of interleukin-1 beta (IL-1 beta), a cytokine that induces bone resorption, to modulate PDGF binding and PDGF-induced biological activity. IL-1 beta significantly reduced PDGF-AA binding and significantly decreased both PDGF-AA-mediated cell migration and thymidine incorporation. In contrast, IL-1 beta had only a small effect of PDGF-BB binding and PDGF-BB-induced biological activity in normal human osteoblastic cells.

Polypeptide growth factors are a class of potent natural biologic mediators which regulate many of the activities of wound healing including cell proliferation, migration, and metabolism. Platelet-derived growth factor (PDGF) and insulin-like growth factor-I (IGF-I) have been shown to regulate DNA and protein synthesis in bone cells in vitro and to interact synergistically to enhance soft tissue wound healing in vivo. We have hypothesized that the combination of PDGF and IGF-I may, therefore, enhance regeneration of both the soft and hard tissue components of the periodontium. To test this hypothesis we performed conventional periodontal surgery on all 4 quadrants of the mouth of 13 beagle dogs with naturally occurring periodontal disease. Following flap reflection, degranulation, and root planing, all premolar teeth in 2 quadrants of each dog received a combination of 3 micrograms of recombinant PDGF-B and IGF-I in a methylcellulose gel, while the premolar teeth in the contralateral quadrants received the gel alone. Teeth in 4 additional animals also received 125I-PDGF or 125I-IGF-I in the treated sites. The clearance rate of the 125I-labeled protein, changes in local bone metabolism, and amount of new bone and cementum with inserting collagen fibers were measured. The clearance studies revealed that the half-life of the factors at the site of application was 3.0 hours for IGF-I and to 4.2 hours for PDGF-B. Greater than 96% of the radio-labeled proteins was cleared by 96 hours and no radioactivity was detected 2 weeks after application. There was a significant (P less than 0.01) 2-fold increase in uptake of the bone-seeking radiopharmaceutical Technetium 99-MDP at 2 and 4 weeks in growth factor treated sites compared to controls, indicating that there was increased metabolic activity within the bone at these sites. Computer-aided histologic analyses of biopsies obtained at 2 and 5 weeks post-operatively revealed a significant (P less than 0.01), 5 to 10 fold increase in new bone and cementum in PDGF-B/IGF-I treated sites at both time points compared to controls receiving the placebo gel. The height and total area of new bone continued to increase from 2 to 5 weeks. The new bone underwent a normal maturation process as judged by histologic appearance. A physiologic periodontal ligament space was also formed between the new bone and new cementum. There was no increase in ankylosis in the treated sites.(ABSTRACT TRUNCATED AT 400 WORDS)

Astrocytomas are highly malignant brain tumors and are among the most neovascularized solid tumors. We have investigated the expression of the angiogenic growth factors acidic fibroblast growth factor and transforming growth factor-alpha, together with its receptor epidermal growth factor receptor, in 30 primary astrocytomas. Both acidic fibroblast growth factor and transforming growth factor-alpha, together with epidermal growth factor receptor, are found to be greatly overexpressed in these tumors when compared with normal brain. This overexpression of angiogenic growth factors may underlie the intense neovascularization characteristic of astrocytomas.

Platelet-derived growth factor (PDGF) stimulates many of the processes important in tissue repair, including proliferation of fibroblasts and synthesis of extracellular matrices. In this study we have demonstrated with in situ hybridization and immunocytochemistry the reversible expression of c-sis/PDGF-2 and PDGF receptor (PDGF-R) b mRNAs and their respective protein products in epithelial cells and fibroblasts following cutaneous injury in pigs. Epithelial cells in control, unwounded skin did not express c-sis and PDGF-R mRNAs, and fibroblasts expressed only PDGF-R mRNA. The expression levels in the injured site were correlated with the stage of tissue repair, being highest during the initial stages of the repair process and declining at the time of complete re-epithelialization and tissue remodeling. It is suggested that the controlled, reversible expression of a potent mitogen and its receptor induced by injury may function in an autocrine/paracrine manner on both epithelial cells and fibroblasts to bring about their sustained proliferation during the normal healing process. These studies provide a molecular basis for understanding the mechanisms contributing to normal tissue repair. We suggest the possibility that a defect in these mechanisms may be associated with defective wound healing. It is also conceivable that "chronic" injury may induce irreversible gene expression leading to pathologic, unregulated cell growth.

Fibrosis is a complex process involving an inflammatory reaction, fibroblast proliferation, and abnormal accumulation of interstitial collagens. Mononuclear cells are usually present in lung fibrosis. Activated monocytes and macrophages in culture have been shown to produce several growth factors including platelet-derived growth factor (PDGF). PDGF is a potent mitogen and chemoattractant for fibroblasts and smooth muscle cells and a stimulator of collagen synthesis. We have studied the expression of c-sis/PDGF-2 mRNA in lung tissues derived from five patients with idiopathic pulmonary fibrosis (IPF) and from four control individuals without IPF. Northern blot analysis of specimens obtained from four patients with IPF revealed the expression of the c-sis/PDGF-2 protooncogene. A control lung tissue without IPF did not express the c-sis protooncogene. In situ hybridization extended these studies demonstrating the expression of the c-sis mRNA in the five specimens with IPF but not in the four control specimens without IPF. The expression of c-sis mRNA was localized primarily in the epithelial cells. Invading alveolar macrophages also expressed c-sis mRNA. The expression of c-sis mRNA was accompanied by the expression of PDGF-like proteins in lung specimens with IPF but not in control lung specimens. These findings demonstrate the in vivo expression of the c-sis/PDGF-2 protooncogene and the production of PDGF-like proteins in the epithelial cells and macrophages of the fibrotic tissue. This localized and sustained production of PDGF-like mitogen may constitute an important contributing factor in the abnormal fibroblast proliferation and collagen production, events associated with pulmonary fibrosis.

The present studies investigated the expression of the platelet-derived growth factor (PDGF) and PDGF-receptor genes in human meningiomas. Northern blot analysis demonstrated that all meningiomas examined expressed both the c-sis/PDGF-2 proto-oncogene and the PDGF-receptor gene. In situ hybridization localized the c-sis mRNA and the PDGF-receptor mRNA in the tumor cells of the meningioma tissues. Control pachymeninges derived from adult individuals, without meningiomas, expressed only PDGF-receptor mRNA but not the c-sis mRNA. Immunocytochemistry studies detected both the c-sis and the PDGF-receptor protein products in meningioma tissues but only the PDGF-receptor protein products in control pachymeninges. These findings indicate the presence of an autocrine mechanism in human meningiomas based on the co-expression of the c-sis/PDGF-2 proto-oncogene and PDGF-receptor gene and their protein products. This co-expression of a potent mitogen and its receptor may contribute to the growth and maintenance of human meningiomas.

The present studies investigated the expression of the two PDGF genes (c-sis/PDGF-2 and PDGF-1) and the PDGF-receptor b gene (PDGF-R) in 34 primary human astrocytomas. Northern blot analysis demonstrated the coexpression of the c-sis/PDGF-2 protooncogene and the PDGF-R gene in all astrocytomas examined. The majority of the tumors also expressed the PDGF-1 gene. There was no correlation between the expression of the two PDGF genes. Nonmalignant human brain tissue expressed the PDGF-R and PDGF-1 genes but not the c-sis/PDGF-2 protooncogene. In situ hybridization of astrocytoma tissue localized the expression of the c-sis and PDGF-R mRNA's in tumor cells. Capillary endothelial cells also expressed c-sis mRNA. In contrast, nonmalignant human brain tissue expressed only PDGF-R mRNA but not c-sis/PDGF-2 mRNA. The coexpression of a potent mitogenic growth factor protooncogene (c-sis) and its receptor gene in astrocytoma tumor cells suggests the presence of an autocrine mechanism that may contribute to the development and maintenance of astrocytomas. The expression of c-sis mRNA in tumor cells but not in nonmalignant brain cells may serve as an additional diagnostic criterion for the detection of astrocytomas in small tissue specimen using in situ hybridization for the detection of c-sis mRNA and/or immunostaining for the recognition of its protein product.

We report that human breast cancer cells secrete a growth factor that is biologically and immunologically similar to platelet-derived growth factor (PDGF). Serum-free medium conditioned by estrogen-independent MDA-MB-231 or estrogen-dependent MCF-7 cells contains a mitogenic or "competence" activity that is capable of inducing incorporation of [3H]thymidine into quiescent Swiss 3T3 cells in the presence of platelet-poor plasma. In addition, the conditioned medium contains an activity that competes with 125I-labeled PDGF for binding to PDGF receptors on normal human fibroblasts. The secretion of PDGF-like activity by the hormone-responsive cell line MCF-7 is stimulated by 17 beta-estradiol. Like authentic PDGF, the PDGF-like activity produced by breast cancer cells is stable after acid and heat treatment (95 degrees C) and inhibited by reducing agents. The mitogenic activity comigrates with a material of approximately equal to 30 kDa on NaDodSO4/polyacrylamide gels. Immunoprecipitation with PDGF antiserum of proteins from metabolically labeled cell lysates and conditioned medium followed by analysis on nonreducing NaDodSO4/polyacrylamide gels identified proteins of 30 and 34 kDa. Upon reduction, the 30- and 34-kDa bands were converted to 15- and 16-kDa bands suggesting that the immunoprecipitated proteins were made up of two disulfide-linked polypeptides similar to PDGF. Hybridization studies with cDNA probes for the A chain of PDGF and the B chain of PDGF/SIS identified transcripts for both PDGF chains in the MCF-7 and MDA-MB-231 cells. The data summarized above provide conclusive evidence for the synthesis and hormonally regulated secretion of a PDGF-like mitogen by breast carcinoma cells. Production of a PDGF-like growth factor by breast cancer cell lines may be important in mediating paracrine stimulation of tumor growth.

Cells derived from the endothelium of human iliac arteries were cultured in vivo. The cells were isolated, grown, and subcultured in HEPES buffered Medium 199 supplemented with 20% heat inactivated human whole blood serum, human alpha-thrombin, and commercial endothelial cell growth supplement derived from bovine brain. The cells were viable in culture for 8 to 10 passages at a split ratio of 1:3. After the 10th passage, the cells began to enlarge and their growth rate was reduced. No cultures were viable after the 12th passage. The cells were determined to be of endothelial origin by their morphology at confluence; their ultrastructural characteristics, including the presence of Weibel-Palade bodies; the production and release of factor VIII-related antigen; and by their maintenance of a surface that prevented platelet attachment. The cultured arterial endothelial cells released prostacyclin in response to challenge with thrombin and protamine sulfate but not in response to bradykinin or the platelet-derived growth factor. Although the cultures described in this report were derived from patients with varying degrees of atherosclerotic disease, there were no significant differences in morphological or physiological parameters among these cultures or in comparison with commonly studied cells derived from human umbilical veins.

U-2 OS human osteosarcoma cells synthesize, process and secrete a platelet-derived growth factor (PDGF)-like mitogen. Incubation of these cells with 1 mM suramin unmasks PDGF receptor sites which are normally occupied or down regulated by the secreted endogenous PDGF-like mitogen. Partially purified preparations of metabolically labelled U-2 OS conditioned medium binds to U-2 OS cells and binding is inhibited by excess PDGF. These findings suggest that U-2 OS cells are capable of autocrine stimulation.

Human malignant epithelial cell lines were analyzed for expression of platelet-derived growth factor (PDGF) genes. Of the 12 cell lines tested, 9, derived from breast, lung, gastric, and ovarian carcinomas, were found to express both PDGF-1 and PDGF-2 genes. The levels of both PDGF-1 and PDGF-2 transcripts were superinduced when these cells were treated with cycloheximide, an inhibitor of protein synthesis. These cells also released an activity that in studies with BALB-c/3T3 cells, inhibited binding of 125I-labeled PDGF and stimulated incorporation of [3H]thymidine. This stimulating activity was inhibited after reduction of the conditioned media by mercaptoethanol or after preincubation with antibodies to PDGF. Moreover, this activity was not affected by heat treatment. Immunoprecipitation studies revealed that breast, lung, and gastric carcinoma cells produced PDGF-like proteins that migrated as 30- and 32-kD species under nonreducing conditions and as 15- and 16-kD species under reducing conditions. In contrast, malignant cells of ovarian origin produced 14-16-kD PDGF-like proteins that were unchanged in mobility after reduction. As PDGF receptors were not detected on these malignant epithelial cells, the production of PDGF-like proteins may affect other cells in the microenvironment by paracrine mechanisms and may contribute to excessive cell proliferation, inflammatory reactions, and connective tissue remodeling seen in certain carcinomas.

Prostacyclin (PGI2), an unstable metabolite of arachidonic acid synthesized by vascular endothelial and smooth muscle cells, is a potent vasodilator and endogenous inhibitor of platelet aggregation. Regulation of PGI synthesis by the vessel wall is not well understood. We have investigated the possibility that a product released from platelet granules during degranulation might modify vessel wall PGI2 biosynthesis. We report here that a non-dialysable, platelet-dependent factor in serum dramatically stimulates PGI2 synthesis by cultured bovine aortic endothelium aortic smooth muscle, and adrenal capillary endothelium. Platelet-derived growth factor (PDGF), a releasable peptide contained within platelet alpha granules, stimulates PGI2 synthesis by the above cell types as much as 100-fold. The concentrations of PDGF required to produce these effects are below the level reported in normal human serum. We postulate that in vivo released PDGF may increase vessel wall PGI2 production as part of a negative feedback mechanism controlling platelet aggregation.

Prostate carcinoma cell lines DU-145 and PC-3 express both platelet derived growth factor (PDGF)-1 and PDGF-2/sis genes. Concomitantly, these cells synthesize and secrete PDGF-like proteins, as judged by indirect immunofluorescence and by direct immunoprecipitation with specific PDGF antiserum. Conditioned media derived from DU-145 and PC-3 cells stimulated the incorporation of [3H]thymidine by 3T3 cells and competed with 125I-labeled PDGF for its binding to cell surface receptors of 3T3 cells. The biological activity was stable to heating at 100 degrees C for 10 min, sensitive to reducing agents, and neutralized by the IgG fraction of PDGF antiserum, properties similar to those of authentic PDGF. Both DU-145 and PC-3 cell lines appear to lack receptors for PDGF as indicated by their inability to mitogenically respond to PDGF and receptor binding of 125I-labeled PDGF. Production of PDGF-like proteins by human prostate carcinoma cells may play an important role in a paracrine mode in the organization of the extracellular matrix of the malignant tissue.

Pure human platelet-derived growth factor at nanogram levels stimulates cholesterol ester, phospholipid and DNA synthesis in normal and familial hypercholesterolemia mutant human skin fibroblasts. Stimulation of DNA synthesis did not begin until 15-24 h after addition of platelet-derived growth factor to quiescent normal and FH mutant fibroblasts, In contrast, stimulation of [3H]oleic acid incorporation into cholesterol ester and phospholipid was evident 3-6 h after the addition of platelet-derived growth factor. In the normal cells, the rate of cholesterol ester synthesis was maximal at 24 h, then rapidly declined. Compared to the normal cells, cholesterol esterification was much lower in the FH cells; however, platelet-derived growth factor stimulated the rate of [3H]oleic acid incorporation into cholesterol ester by 5-fold, 31 h after addition of the growth factor. The stimulation of [3H]oleic acid incorporation into cholesterol ester by platelet-derived growth factor was inhibited in both normal and FH mutant skin fibroblasts by progesterone, an inhibitor of acyl-CoA: cholesterol acyltransferase. The rate of cholesterol ester synthesis in the normal cells increased as the concentration of platelet-poor plasma or low density lipoprotein (LDL) was increased, especially in the presence of platelet-derived growth factor. Linearization of the LDL dose-response curve indicated that platelet-derived growth factor increased the rate rather than the affinity of the overall cholesterol esterification system. The rate of cholesterol esterification in the FH mutant cells was highest in the absence of LDL or at low levels of platelet-poor plasma. Consequently, platelet-derived growth factor can stimulate cholesterol ester synthesis by LDL- and non-LDL-mediated processes.

Quiescent BALB/c 3T3 cells exposed briefly to a platelet-derived growth factor (PDGF) become "competent" to replicate their DNA but do not "progress" into S phase unless incubated with growth factors contained in platelet-poor plasma. Plasma from hypophysectomized rats is deficient in progression activity; it does not stimulate PDGF-treated competent cells to synthesize DNA, demonstrating that somatomedin C is required for progression. Various growth factors were tested for progression activity and competence activity by using BALB/c 3T3 tissue culture assays. Multiplication stimulating activity and other members of the somatomedin family of growth factors are (like somatomedin C) potent mediators of progression. Other mitogenic agents, such as fibroblast growth factor, are (like PDGF) potent inducers of competence. Growth factors with potent progression activity have little or no competence activity and vice versa. In contrast, simian virus 40 provides both competence and progression activity. Coordinate control of BALB/c 3T3 cell growth in vitro by competence factors and somatomedins may be a specific example of a common pattern of growth regulation in animal tissues.

Serum contains a growth factor derived from platelets and also growth factors derived from platelet-poor plasma. Extracts of heated (100 degrees ) human platelets function synergistically with platelet-poor plasma to induce DNA synthesis in quiescent, density-inhibited BALB/c 3T3 cells. Platelet-poor plasma alone did not induce DNA synthesis. Cells exposed to platelet extracts became competent to enter the cell cycle, but the rate of entry into the S phase depended upon the concentration of platelet-poor plasma. The time required for the induction of this competent state was a function of the concentration of the platelet extract. A 2-hr exposure to 100 mug of the platelet extract at 37 degrees caused the entire cell population to become competent to enter the S phase. At 4 degrees or 25 degrees the cells did not become competent to synthesize DNA. The platelet extract-induced competent state was stable for at least 13 hr after removal of the platelet extract; however, in the absence of platelet-poor plasma, these competent cells did not progress through the cell cycle. The addition of an optimal concentration of platelet-poor plasma (5%) to these competent cells initiated cell cycle traverse with a rapid, first-order entry of cells into the S phase beginning 12 hr after addition of the plasma. The addition of a suboptimal concentration of the plasma (0.25%) did not increase the rate of cell entry into the S phase. Thus, the induction of DNA synthesis in quiescent BALB/c 3T3 cells can be resolved into at least two phases, controlled by different serum components: (i) competence, induced by the platelet-derived growth factor; and (ii) progression of competent cells into the cell cycle, mediated by factors in platelet-poor plasma.

The addition of whole serum to G0-arrested, confluent Balb/c-3T3 cells induces them to progress through G1 and synthesize DNA after a 12-h lag period. Prior to the onset of DNA synthesis, RNA is synthesized and RNA content increases. Serum has been fractionated into two sets of growth factors: a platelet-derived growth factor present in heat-treated (100 degrees C) platelet extracts and platelet-poor plasma. Addition of whole serum, platelet-derived growth factor or platelet-poor plasma induces quiescent cells to increase their cytoplasmic RNA content, but the cells treated with platelet-poor plasma do not synthesize DNA. Messenger RNA content increases within 2 h after stimulation with whole serum or platelet-poor plasma, and after 18 h, mRNA has accumulated to a greater degree than rRNA.

In human umbilical vein and bovine aortic endothelial cells in culture c-sis gene expression and secretion of platelet-derived growth factor (PDGF) has been previously demonstrated. We now report the presence of PDGF-1 and PDGF-2/sis mRNA transcripts in primary cultures of human iliac artery endothelial cells (HIA-EC). Concomitantly, these cells synthesize and secrete PDGF-like proteins identified by direct immunoprecipitation with specific PDGF antiserum. The PDGF proteins secreted by HIA-EC have molecular weights of 31 and 35 kd under nonreducing conditions. Upon reduction these proteins are converted to the monomeric 15- and 16-kd forms. Conditioned media derived from HIA-EC stimulated the incorporation of 3H-thymidine by 3T3 cells and competed with 125I-PDGF for its binding to 3T3 cell membrane receptors. The biologic activity was stable to heating at 100 degrees C for 10 min and sensitive to reducing agents, properties similar to those of authentic PDGF. Production of PDGF-like mitogen by the human arterial endothelial cells may play an important role in the paracrine modulation of arterial wall regeneration following vascular injury.

A basic polypeptide that stimulates DNA synthesis and cell division in confluent populations of mouse Balb/c-3T3 cells has been isolated from whole human serum, and has been separated from the heterogenous group of molecules with insulin-like activity. This highly purified basic polypeptide has a molecular weight of 1.3 x 10(4) and an isoelectric point of 9.7. Approximately 10(7) polypeptide molecules in the growth medium allow the replication of one density-inhibited cell.

A new class of polypeptide hormones known collectively as growth factors has been identified. These polypeptides are able to stimulate DNA synthesis and mitosis of cells cultured in vitro. Growth factors have been isolated from several sources, including platelets, submaxillary glands, pituitary, brain, and medium conditioned by cells grown in vitro. Growth factors appear to behave like classic polypeptide hormones. In the cases of epidermal growth factor and nerve growth factor, specific cell membrane receptors have been studied in detail and partially purified. Platelet-derived growth factor and somatomedin-C interact synergistically and apparently, sequentially to promote cell proliferation in vitro. These studies suggest that cell proliferation is controlled by a synergistic interaction among several growth factors and, perhaps, other hormones. Several in vivo roles for growth factors and a role for tumor-cell-derived growth factors in neoplasia have been suggested.

Normal rat kidney (NRK) cells transformed by simian sarcoma virus (SSV) release into the culture medium a biologically active mitogen with properties identical to those of human platelet-derived growth factor (PDGF). Like PDGF, the growth factor derived from SSV-NRK cells was shown to be stable to heat and sensitive to reducing agents. It was capable of inhibiting binding of labeled PDGF to the receptor on human fibroblasts. It also stimulated the phosphorylation of the same membrane protein (185 kilodaltons) in isolated plasma membranes from human fibroblasts. Immunoprecipitation of metabolically labeled proteins released by SSV-NRK cells showed that a 34-kilodalton protein was specifically precipitated by antiserum to PDGF. Upon reduction, this protein had a molecular size of 17 kilodaltons. PDGF has been shown to consist of two 14- to 18-kilodalton proteins linked by disulfide bonds.