Vascular permeability factor (vascular endothelial growth factor) expression and angiogenesis in cervical neoplasia.

BACKGROUND: Angiogenesis is a critical factor in the progression of solid tumors, including cervical cancers. The mechanisms responsible for angiogenesis in cervical neoplasia, however, are not well defined. PURPOSE: Our goal was to determine the relationship between angiogenesis and the expression of the angiogenic cytokine vascular permeability factor (VPF), also known as vascular endothelial growth factor, and its receptors in cervical neoplasia. METHODS: Sixty-six cervical biopsy specimens were evaluated; among these, 16 samples were designated as benign, 17 as low-grade squamous intraepithelial lesions, 18 as high-grade squamous intraepithelial lesions, and 15 as invasive squamous cell carcinomas. Histologic sections immunostained for factor VIII-related antigen were evaluated quantitatively for microvessel density and for the presence of epithelial-stromal vascular cuffing. Sections were also evaluated for VPF messenger RNA (mRNA) expression by in situ hybridization. RESULTS: VPF mRNA expression, epithelial-stromal vascular cuffing, and microvessel density counts were significantly increased in invasive carcinoma and in high-grade intraepithelial lesions as compared with low-grade intraepithelial lesions and benign squamous epithelium. Vascular cuffing and increased microvessel density counts were also significantly associated with increased VPF mRNA expression. CONCLUSIONS: These observations suggest that VPF is an important angiogenic factor in cervical neoplasia.

Expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in adenocarcinomas of the gastrointestinal tract.

Vascular permeability factor (VPF) is one of the most potent known inducers of microvascular hyperpermeability; in addition, it is a selective endothelial cell growth factor, hence its alternate name, vascular endothelial growth factor. VPF exerts its actions on the microvasculature by interacting with specific endothelial cell receptors. VPF is expressed by many transplantable animal tumors, by tumor cell lines in culture, and by certain normal cells in situ. The purpose of the present investigation was to determine whether and with what consistency VPF and its endothelial cell receptors are expressed in primary autochthonous human tumor of gastrointestinal tract origin, as determined by in situ hybridization and immunohistochemistry. Twenty-one primary adenocarcinomas (17 colon, 2 stomach, 1 small bowel, and 1 pancreas) were studied. The malignant epithelial cells expressed VPF mRNA strongly, in contrast to normal epithelium, hyperplastic polyps, and adenomas, which expressed little or no VPF mRNA. VPF expression was further increased in tumor cells immediately adjacent to zones of tumor necrosis; in such areas, occasional stromal cells also expressed VPF mRNA. In the ten colon carcinomas studied, tumor cells stained for VPF protein by immunohistochemistry. The endothelial cells of nearby stromal blood vessels also stained for VPF by immunohistochemistry and in addition expressed mRNAs encoding the VPF receptors flt-1 and kdr as determined by in situ hybridization. Endothelial cells away from the tumor did not stain for VPF and no definite mRNA expression for flt-1 or kdr was detected by in situ hybridization. The ganglion cells of the myenteric plexus of normal bowel expressed VPF mRNA and protein. These data indicate that primary autochthonous human tumors of gastrointestinal origin regularly express both VPF mRNA and VPF protein and that adjacent stromal vessels express mRNAs for both known VPF receptors. VPF is likely to contribute to tumor growth by promoting angiogenesis and stroma formation, both directly, through its action as an endothelial cell growth factor, and indirectly, by increasing vascular permeability, thereby leading to plasma protein extravasation, fibrin deposition, and the eventual replacement of the resulting matrix with vascularized stroma.

Expression of vascular permeability factor/vascular endothelial growth factor by melanoma cells increases tumor growth, angiogenesis, and experimental metastasis.

Vascular permeability factor (VPF)/vascular endothelial growth factor (VEGF) is an angiogenic cytokine expressed by many human and animal tumors. Hypoxia often up-regulates VPF/VEGF expression further. To better define the role of VPF/VEGF in tumor biology, we screened tumorigenic lines for those expressing minimal constitutive and hypoxia-inducible VPF/VEGF. Human melanoma SK-MEL-2 cells best fit these criteria and formed small, poorly vascularized tumors in immunodeficient mice. We transfected SK-MEL-2 cells stably with sense or antisense mouse VPF/VEGF cDNA or with vector alone. Cells transfected with sense VPF/VEGF (V+) expressed and secreted large amounts of mouse VPF/VEGF and formed well-vascularized tumors with hyperpermeable blood vessels and minimal necrosis in nude/SCID mice. Antisense-transfected VPF/VEGF (V-) cells expressed reduced constitutive VPF/VEGF and no detectable mouse VPF/VEGF, and formed small, minimally vascularized tumors exhibiting extensive necrosis. Vector-alone transfectants (N1 cells) behaved like parental cells. V+ cells formed numerous lung tumor colonies in SCID mice, approximately 50-fold more than N1 cells, whereas V- cells formed few or none. These experiments demonstrate that VPF/VEGF promotes melanoma growth by stimulating angiogenesis and that constitutive VPF/VEGF expression dramatically promotes tumor colonization in the lung.

Vascular stroma formation in carcinoma in situ, invasive carcinoma, and metastatic carcinoma of the breast.

The generation of vascular stroma is essential for solid tumor growth and involves stimulatory and inhibiting factors as well as stromal components that regulate functions such as cellular adhesion, migration, and gene expression. In an effort to obtain a more integrated understanding of vascular stroma formation in breast carcinoma, we examined expression of the angiogenic factor vascular permeability factor (VPF)/vascular endothelial growth factor (VEGF); the VPF/VEGF receptors flt-1 and KDR; thrombospondin-1, which has been reported to inhibit angiogenesis; and the stromal components collagen type I, total fibronectin, ED-A+ fibronectin, versican, and decorin by mRNA in situ hybridization on frozen sections of 113 blocks of breast tissue from 68 patients including 28 sections of breast tissue without malignancy, 18 with in situ carcinomas, 56 with invasive carcinomas, and 8 with metastatic carcinomas. A characteristic expression profile emerged that was remarkably similar in invasive carcinoma, carcinoma in situ, and metastatic carcinoma, with the following characteristics: strong tumor cell expression of VPF/VEGF; strong endothelial cell expression of VPF/VEGF receptors; strong expression of thrombospondin-1 by stromal cells and occasionally by tumor cells; and strong stromal cell expression of collagen type I, total fibronectin, ED-A+ fibronectin, versican, and decorin. The formation of vascular stroma preceded invasion, raising the possibility that tumor cells invade not into normal breast stroma but rather into a richly vascular stroma that they have induced. Similarly, tumor cells at sites of metastasis appear to induce the vascular stroma in which they grow. We conclude that a distinct pattern of mRNA expression characterizes the generation of vascular stroma in breast cancer and that the formation of vascular stroma may play a role not only in growth of the primary tumor but also in invasion and metastasis.

Increased expression of vascular permeability factor (vascular endothelial growth factor) in bullous pemphigoid, dermatitis herpetiformis, and erythema multiforme.

Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), plays an important role in the increased vascular permeability and angiogenesis associated with many malignant tumors. In addition, VPF/VEGF is strongly expressed by epidermal keratinocytes in wound healing and psoriasis, disorders that are also characterized by increased microvascular permeability and angiogenesis. In this study, we investigated the expression of VPF/VEGF in three bullous diseases with subepidermal blister formation that are characterized by hyperpermeable dermal microvessels and pronounced papillary dermal edema. The expression of VPF/VEGF mRNA was strongly up-regulated in the lesional epidermis of bullous pemphigoid (n = 3), erythema multiforme (n = 3), and dermatitis herpetiformis (n = 4) as detected by in situ hybridization. Epidermal labeling was particularly intense over blisters, but strong expression was also noted in areas of the epidermis adjacent to dermal inflammatory infiltrates at a distance from blisters. Moreover, the VPF/VEGF receptors, flt-1 and KDR, were up-regulated in endothelial cells in superficial dermal microvessels. High levels of VPF/VEGF (138-238 pM) were detected in blister fluids obtained from five patients with bullous pemphigoid. Addition of blister fluid to human dermal microvascular endothelial cells exerted a dose-dependent mitogenic effect that was suppressed after depletion of VPF/VEGF by immunoadsorption. These findings strongly suggest that VPF/VEGF plays an important role in the induction of increased microvascular permeability in bullous diseases, leading to papillary edema and fibrin deposition and contributing to the bulla formation characteristic of these disorders.

Vascular endothelial growth factor-C (VEGF-C) and its receptors KDR and flt-4 are expressed in AIDS-associated Kaposi's sarcoma.

Kaposi's sarcoma is characterized by clusters of spindle-shaped cells that are considered to be tumor cells and by prominent vasculature. Whereas spindle cells are most likely endothelial in origin, it remains controversial whether they are of lymphatic or blood vascular derivation. To test the hypothesis that the lymphangiogenesis factor vascular endothelial growth factor-C and its receptors, KDR and flt-4, are involved in the pathogenesis of Kaposi's sarcoma, we performed in situ hybridizations and immunofluorescent stainings on human immunodeficiency virus-associated Kaposi's sarcoma. Spindle-shaped tumor cells strongly expressed KDR and flt-4 mRNA. Immunofluorescent staining confirmed expression of the flt-4 receptor in Kaposi's sarcoma cells, and double labeling revealed its colocalization with the endothelial cell marker CD31. Vascular endothelial growth factor-C was strongly expressed in blood vessels associated with Kaposi's sarcoma. In vitro, human dermal microvascular endothelial cells also expressed vascular endothelial growth factor-C mRNA that was further upregulated by vascular permeability factor/vascular endothelial growth factor. Vascular endothelial growth factor-C potently stimulated the proliferation of Kaposi's sarcoma tumor cells in vitro. These results demonstrate important paracrine functions of vascular endothelial growth factor-C, produced by blood vessels, in the pathogenesis of cutaneous Kaposi's sarcoma, and suggest a lymphatic origin and/or differentiation of Kaposi's sarcoma tumor cells.

Cloning of DLM-1, a novel gene that is up-regulated in activated macrophages, using RNA differential display.

Tumors interact with their environment, reprogramming host cells to induce responses such as angiogenesis, inflammation, immunity and immune suppression. To understand these processes, it is important to identify and isolate new genes whose expression is induced in host tissues in response to tumors. Ascites tumors offer an attractive model for isolating such genes, because responding host peritoneal lining tissues can be cleanly separated from tumor cells growing in suspension within the peritoneal cavity. We here report the cloning by differential display of a novel gene, DLM-1, that is highly up-regulated in the peritoneal lining tissue of mice bearing MOT ascites tumors. Mouse peritoneal macrophages, stimulated by IFN-gamma or LPS, also expressed significant amounts of DLM-1. Up-regulation of DLM-1 became evident by 4h after stimulation with IFN-gamma and was not blocked by cycloheximide, suggesting the presence of IFN responding elements in its transcription regulation region. DLM-1 RNA was detected at significant levels in normal mouse lung, intestinal epithelium, liver and thymus by Northern blot analysis. In situ hybridization of MOT and HT-29 mouse subcutaneous transplanted solid tumors revealed strong DLM-1 expression in the host reactive stromal cells, but not the tumor cells. Sequence analysis of the full-length cDNA clone revealed that it encodes a protein of approx. M(r) 44330 with multiple potential protein kinase C and casein kinase II phosphorylation sites. Our data suggest that DLM-1 plays a role in such important processes as host response in neoplasia.

Vascular permeability factor/vascular endothelial growth factor and vascular stroma formation in neoplasia. Insights from in situ hybridization studies.

The formation of vascular stroma plays an important role in the pathophysiology of malignancy. We describe the use of in situ hybridization in our laboratory as a tool to study the role of vascular permeability factor/vascular endothelial growth factor in the angiogenesis associated with malignancy.

Expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in breast cancer.

Solid tumors must induce a vascular stroma to grow beyond a minimal size, and the intensity of the angiogenic response has been correlated with prognosis in breast cancer patients. Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), is a secreted protein that has been implicated in tumor-associated angiogenesis. Vascular permeability factor directly stimulates endothelial cell growth and also increases microvascular permeability, leading to the extravasation of plasma proteins, which alter the extracellular matrix in a manner that promotes angiogenesis. To determine whether VPF has a role in breast cancer, we used in situ hybridization to study VPF mRNA expression in normal breast tissue (13 specimens), comedo-type ductal carcinoma in situ (DCIS) (four specimens), infiltrating ductal carcinoma (12 specimens), infiltrating lobular carcinoma (two specimens), metastatic ductal carcinoma (three specimens) and metastatic lobular carcinoma (one specimen). Vascular permeability factor mRNA was expressed at a low level by normal duct epithelium but was expressed at high levels in tumor cells in all cases of comedo-type DCIS, infiltrating ductal carcinoma, and metastatic ductal carcinoma. In contrast, VPF mRNA was not expressed at high levels in infiltrating lobular carcinoma. We also used in situ hybridization to study the expression of two recently described endothelial cell surface VPF receptors, flt-1 and kdr. Vascular permeability factor receptor mRNA was strongly expressed in endothelial cells of small vessels adjacent to malignant tumor cells in DCIS, infiltrating ductal carcinoma, and metastatic ductal carcinoma. In contrast, no definite labeling for receptor mRNA was detected in infiltrating lobular carcinoma or nonmalignant breast tissue. The intense expression of VPF mRNA by breast carcinoma cells and of VPF receptor mRNA by endothelial cells of adjacent small blood vessels provides strong evidence linking VPF expression to the angiogenesis associated with comedo-type DCIS, infiltrating ductal, and metastatic ductal breast carcinoma.

Identification and partial characterization of PDZK1: a novel protein containing PDZ interaction domains.

We recently reported the isolation and partial characterization of a novel membrane-associated protein designated MAP17. In normal tissues, MAP17 was expressed only in the apical brush border of proximal tubular epithelial cells of the human adult kidney. However, MAP17 was diffusely expressed in most carcinomas originating in the kidney, colon, lung, and breast. Transfection of MAP17 into the HT29 carcinoma cell line markedly decreased cell proliferation in vitro and tumor growth in vivo, suggesting that MAP17 plays a role, either direct or indirect, in the control of cell proliferation. In an attempt to elucidate the function of MAP17, we screened a human kidney cDNA library for interacting proteins using the yeast two-hybrid system and isolated a novel protein containing PDZ protein interaction domains, which we have named PDZK1. PDZK1 is a 519-amino acid protein with a molecular weight of 63 kd; it is expressed in the kidney, pancreas, liver, gastrointestinal tract, and adrenal cortex. In situ hybridization experiments showed that the expression of PDZK1 was limited to epithelial cells. In the kidney, it colocalized with MAP17 in the brush border of proximal tubular epithelial cells. In addition, PDZK1 was overexpressed in selected tumors of epithelial origin. Although the function of PDZK1 has yet to be determined, proteins containing PDZ domains have been shown to play important roles as diverse as cell-cell interaction, cell differentiation, growth control, ion channels organization, and signal transduction. This is of particular interest because MAP17 is localized in areas either of cell-cell contact or where ion channels are localized, for example in the kidney. PDZK1 may represent the link between the cell membrane-where it interacts with MAP17-and other cytoplasmic proteins involved in biologic functions such as cell proliferation, differentiation, and ion transport.

Osteopontin is strongly expressed by histiocytes in granulomas of diverse etiology.

Granulomatous inflammation is associated with a variety of important pathologic conditions. Osteopontin (OPN), a ligand for the alpha v beta 3 integrin, is a secreted glycoprotein with a glycine-arginine-glycine-aspartate-serine cell-binding domain. In this study, we examined expression of OPN in 22 cases of granulomatous inflammation including cases of sarcoidosis, granulomatous temporal arteritis, histoplasmosis, rheumatoid nodule, granuloma annulare, erythema nodosum, granulomatous gastritis, foreign body giant-cell granulomatous reactions, and lipogranulomas. Strong expression of OPN mRNA and protein was seen in the epithelioid histiocytes and multinucleate histiocytic giant cells in granulomas by in situ hybridization and immunostaining. OPN may play an important role in granulomatous inflammation through the regulation of processes such as histiocyte migration, cell adhesion, and cellular functions including phagocytosis.

Vascular permeability factor/vascular endothelial growth factor and its receptors in oral and laryngeal squamous cell carcinoma and dysplasia.

Increased microvessel density has been described in squamous cell carcinoma of the head and neck and is related to patient prognosis. The factors responsible for the angiogenesis have not been identified. Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), is a multifunctional angiogenic cytokine expressed at high levels in many tumors. We examined 16 cases of squamous cell carcinoma, 10 cases of high-grade squamous dysplasia, and 19 cases of normal, reactive, or mildly dysplastic squamous mucosa by in situ hybridization for expression of VPF/VEGF and VPF/VEGF receptor mRNA. Strong expression of VPF/VEGF mRNA was seen in 12 of 16 squamous cell carcinomas and in 5 of 10 high-grade squamous dysplasias. In contrast, no cases of normal, reactive, or mild dysplastic squamous epithelium showed strong expression of VPF/VEGF mRNA. Furthermore, strong expression of VPF/VEGF receptor mRNA was seen in 5 of 9 cases of squamous cell carcinoma and 3 of 6 cases of high-grade squamous dysplasia but in only 2 of 14 cases of normal, reactive, or mild dysplastic squamous epithelium. Thus, expression of VPF/VEGF and its receptors is markedly increased in high-grade squamous dysplasia and invasive squamous cell carcinoma of the oral cavity and larynx and may play an important role in the angiogenesis associated with these lesions.

Uterine smooth muscle cells express functional receptors (flt-1 and KDR) for vascular permeability factor/vascular endothelial growth factor.

Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), is an angiogenic factor with important roles in tumor growth, wound healing, and inflammation. VPF/VEGF interacts with endothelial cells by way of two high-affinity receptor tyrosine kinases: flt-1 and KDR. The vast majority of published studies have described expression of the VPF/VEGF receptors only in endothelial cells, and the statement is frequently made that these receptors are endothelial-cell-specific. In this study, we detected mRNA for flt-1 and KDR by in situ hybridization in smooth muscle cells in sections of the wall of the uterus. To confirm these unexpected findings, smooth muscle cells from the uterus and, as a control, from the colon were isolated, characterized, and cultured. Both uterine and colonic smooth muscle cells in culture expressed VPF/VEGF, but only smooth muscle cells from the uterus expressed mRNA for flt-1 and KDR by Northern analysis and in situ hybridization. Cell culture extracts of uterine but not colonic smooth muscle cells were also positive for flt-1 by Western analysis. Moreover, cultured uterine but not clonic smooth muscle cells phosphorylated the flt-1 receptor and proliferated strongly in response to added VPF/VEGF. This is one of the first rigorous demonstrations that a normal cell type other than endothelial cells can express functional receptors for VPF/VEGF in vivo and in vitro, suggesting that VPF/VEGF may have important, previously unsuspected roles on cell types other than endothelium.

Expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in endometrial carcinoma.

BACKGROUND: Solid tumors, including endometrial carcinomas, must induce a vascular stroma to grow beyond a minimal size. The mechanisms responsible for angiogenesis in endometrial carcinoma, however, are not well defined. Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), is a multifunctional cytokine that is an important regulator of tumor angiogenesis. We evaluated VPF/VEGF mRNA and protein expression, as well as VPF/VEGF receptor mRNA expression, in endometrial carcinoma. METHODS: Fourteen examples of endometrial carcinoma were evaluated by in situ hybridization; in 7 cases, benign atrophic endometrium from the same patient was also examined. Histologic sections were subjected to in situ hybridization using 35S-labeled riboprobes specific for VPF/VEGF and, in a subset of cases, riboprobes specific for the VPF/VEGF receptors flt-1 and KDR. In addition, ten examples of endometrial carcinoma were evaluated for VPE/VEGF protein expression by immunohistochemistry. RESULTS: All 14 examples of endometrial carcinoma studied by in situ hybridization exhibited focal strong VPF/VEGF mRNA expression by tumor cells. In addition, the endothelial cells of surrounding microvessels strongly expressed flt-1 and KDR mRNAs in all ten cases examined. In contrast, no strong expression of VPF/VEGF, flt-1, or KDR mRNA was observed in the seven examples of benign atrophic endometrium studied. All ten cases of endometrial carcinoma studied by immunohistochemistry exhibited strong VPF/VEGF protein expression by tumor cells. CONCLUSIONS: These observations suggest that VPF/VEGF is an important angiogenic factor in endometrial carcinoma.

Strong expression of kinase insert domain-containing receptor, a vascular permeability factor/vascular endothelial growth factor receptor in AIDS-associated Kaposi's sarcoma and cutaneous angiosarcoma.

Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), plays an important role in the angiogenesis associated with the growth of many human and animal tumors. VPF/VEGF stimulates endothelial cell growth and increases microvascular permeability by interacting with two endothelial cell tyrosine kinase receptors, KDR and flt-1. We studied 16 cases of AIDS-associated Kaposi's sarcoma (KS), 2 cases of cutaneous angiosarcoma, and 6 cases of capillary hemangioma by in situ hybridization for expression of VPF/VEGF, KDR, and flt-1 mRNAs. We also performed immunohistochemical staining for VPF/VEGF protein in 15 cases. Tumor cells in KS and angiosarcoma strongly expressed KDR but not flt-1 mRNA. Endothelial cells in small stromal vessels in and around these tumors strongly expressed both KDR and flt-1 mRNAs. Tumor cells expressed VPF/VEGF mRNA strongly in only one case of KS, adjacent to an area of necrosis. This was also the only case in which the tumor cells stained substantially for VPF/VEGF protein. VPF/VEGF mRNA and protein were, however, strongly expressed by squamous epithelium in areas of hyperplasia and near areas of ulceration overlying tumors. VPF/VEGF mRNA was also expressed focally at lower levels by infiltrating inflammatory cells, probably macrophages. The strong expression of both KDR and flt-1 in small stromal vessels in and around tumors suggests that VPF/VEGF may be an important regulator of the edema and angiogenesis seen in these tumors. The strong expression of KDR by tumor cells in KS and angiosarcoma implies that VPF/VEGF may also have a direct effect on tumor cells. Tumor cells in four of six capillary hemangiomas strongly expressed both KDR and flt-1 mRNAs in contrast to the high level expression of only KDR observed in the malignant vascular tumors studied. Neither VPF/VEGF mRNA or protein were strongly expressed in capillary hemangiomas. VPF/VEGF and its receptors may play an important but as yet incompletely understood role in the pathogenesis of both benign and malignant vascular tumors.

Expression of vascular permeability factor (VPF/VEGF) is altered in many glomerular diseases.

Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), is a potent enhancer of microvascular permeability and a selective endothelial cell growth factor. In normal human kidney, VPF/VEGF mRNA and protein are strongly expressed by visceral glomerular epithelial cells, and VPF/VEGF may be an important regulator of glomerular endothelial cell function. This study examined 47 renal biopsies from patients with a variety of glomerular diseases for expression of VPF/VEGF mRNA and protein by in situ hybridization and immunohisto-chemistry. In many glomerular diseases, VPF/VEGF-expressing cells were decreased in number or absent in areas of focal or global glomerular sclerosis. Decreased numbers of VPF/VEGF-expressing cells in glomeruli were also noted in amyloidosis, diabetes, crescentic glomerulonephritis, and diffuse endocapillary proliferative glomerulonephritis associated with systemic lupus erythematosus. Normally, release of VPF/ VEGF must be under strict control because it is some 50,000 times more potent than histamine as an inducer of microvascular permeability. Damage to visceral epithelial cells in a variety of glomerular diseases has the potential for releasing relatively large amounts of VPF/VEGF locally, leading to increased glomerular permeability. In addition, because VPF/ VEGF is also an endothelial growth factor, the loss of normal, controlled secretion of VPF/VEGF after damage to visceral epithelial cells could lead to important alterations in glomerular endothelial cell function.

Expression of Tie1, Tie2, and angiopoietins 1, 2, and 4 in Kaposi's sarcoma and cutaneous angiosarcoma.

The angiopoietins are recently described growth factors for vascular endothelium. The Tie1 and Tie2 receptors are expressed by endothelium. Acquired immune deficiency syndrome (AIDS)-associated Kaposi's sarcoma (KS) and cutaneous angiosarcoma are malignancies of endothelial origin. KS involves primarily the skin and mucosal surfaces and is common in AIDS patients. In an effort to determine whether the angiopoietins and Tie receptors play a role in the pathobiology of angiosarcoma and KS, we studied the expression of angiopoietin-1, angiopoietin-2, angiopoietin-4, Tie1, and Tie2 mRNAs in biopsies of KS from 12 AIDS patients, in biopsies of cutaneous angiosarcoma from two patients, and in control biopsies of normal skin from three volunteers by in situ hybridization. Strong expression of angiopoietin-2, Tie1, and Tie2 mRNAs was detected in the tumor cells of KS and cutaneous angiosarcomas, in contrast to the focal low-level expression in normal skin biopsies. Focal low-level expression of angiopoietin-1 was seen in KS, cutaneous angiosarcomas, and in normal skin. Focal low-level expression of angiopoietin-4 was identified in a minority of KS lesions. These findings suggest that the angiopoietins and Tie receptors may play an important role in the pathobiology of KS and cutaneous angiosarcoma and identify additional potential targets for therapeutic intervention in these vascular malignancies.

Fibroblast migration in fibrin gel matrices.

In healing wounds and many solid tumors, locally increased microvascular permeability results in extravasation of fibrinogen and its extravascular coagulation to form a fibrin gel, with concomitant covalent cross-linking of fibrin by factor XIIIa. Subsequently, inflammatory cells, fibroblasts, and endothelial cells migrate into the gel and organize it into granulation tissue and later into mature collagenous connective tissue. To gain insight into some of the cell migration events associated with these processes, we developed a quantitative in vitro assay that permits the study of fibroblast migration in fibrin gels. Early passage human or rat fibroblasts were allowed to attach to tissue culture dishes and then were overlaid with a thin layer of fibrinogen that was clotted with thrombin. Fibroblasts began to migrate upwards into the fibrin within 24 hours and their numbers and the distance migrated were quantified over several days. The extent of fibroblast migration was affected importantly by the nature of the fibrin clot. Fibroblasts migrated optimally into gels prepared from fibrinogen at concentrations of -3 mg/ml; ie, near normal plasma fibrinogen levels. Migration was greatly enhanced by extensive cross-linking of the fibrin alpha-chains by factor XIIIa, as occurs when clotting takes place in vivo. When fibrinogen was clotted in Dulbecco's modified Eagle's medium, gamma-chains were cross-linked, but alpha-chain cross-linking was strikingly inhibited, and fibroblasts migrated poorly. Gels prepared from factor XIII-depleted fibrinogen exhibited neither alpha-nor gamma-chain cross-linking and did not support fibroblast migration. Further purification of fibrinogen by anion exchange high pressure liquid chromatography depleted fibrinogen of fibronectin, plasminogen, and other impurities; this purified fibrinogen clotted to form fibrin gels that supported reproducible fibroblast migration.

PDZK1, a novel PDZ domain-containing protein up-regulated in carcinomas and mapped to chromosome 1q21, interacts with cMOAT (MRP2), the multidrug resistance-associated protein.

We recently reported the isolation and partial characterization of two novel proteins, MAP17 and PDZK1. Using in situ hybridization, we demonstrated that MAP17 and PDZK1 mRNAs are markedly up-regulated in human carcinomas. PDZK1, originally isolated as a protein interacting with MAP17, contains four PDZ protein-interaction domains and could potentially interact with as many as four target proteins. In this paper, we confirm the overexpression of PDZK1 in human carcinomas using a specific antibody and demonstrate the localization of the PDZK1 gene to human chromosome 1q21, a region frequently altered in neoplastic conditions. Using the yeast two-hybrid system, we have also determined that PDZK1 interacts with the carboxy-terminal portion of cMOAT (MRP2), the canalicular multispecific organic anion transporter associated with multidrug resistance. This is of particular interest because proteins containing PDZ domains are involved in the clustering and signaling pathways of membrane-associated proteins, including ion channels. Therefore, the protein cluster formed by the association of cMOAT, PDZK1, and MAP17 could play an important role in the cellular mechanisms associated with multidrug resistance, and PDZK1 may represent a new target in cancer cells resistant to chemotherapeutic agents.

Differential expression of frpHE: a novel human stromal protein of the secreted frizzled gene family, during the endometrial cycle and malignancy.

By using the differential display technique to identify genes that are differentially expressed in human endometrial carcinoma compared with normal endometrium, we have cloned frpHE, a novel member of the secreted frizzled gene family. By in situ hybridization, we have determined that frpHE is expressed by mesenchymal cells but not by epithelial cells. The expression of frpHE is modulated during the endometrial cycle: it is expressed in the stroma of proliferative endometrium and not significantly detectable in secretory or menstrual endometrium, suggesting that frpHE is under hormonal regulation. In addition, the expression of frpHE mRNA is markedly up-regulated in the stroma of endometrial hyperplasia and carcinoma and in the stroma of in situ and infiltrating breast carcinomas. Injection of frpHE mRNA in Xenopus embryos inhibited the Wnt-8 mediated dorsal axis duplication. These results indicate that frpHE functions as a regulator of the Wnt-frizzled signaling pathway and is involved in endometrial physiology and carcinogenesis.