Charles River Sprague Dawley rats lack early age-dependent susceptibility to DMBA-induced mammary carcinogenesis.

Developmental stages of mammary glands influence their susceptibility to initiating events related to carcinogenesis. The "window of susceptibility" to mammary carcinogenesis is classically defined as the time in early puberty when the mammary gland morphology is most sensitive to initiation events. Administration of the polyaromatic hydrocarbon, 7,12-dimethylbenz(a)anthracene (DMBA), in a single oral dose yields maximal mammary tumor formation when administered in this "window". We examined the DMBA treated mammary glands, precursor lesions, and morphology of the uninvolved mammary epithelium for the first 100 days of life for Charles River Sprague Dawley CD(R) IGS. Our goal was to determine the DMBA dose at which 50% of the rats (IC50) developed carcinoma in situ (CIS) within three months of dosing. Here we demonstrate, rather than the classical U-shaped dose curve in which there is maximum sensitivity for DMBA at 50 days, there is an increasing degree of sensitivity with age in the CD(R) IGS rat. Additionally, we report that vehicle-treated animals developed mammary CIS without any known initiator, and 100 day virgin animals demonstrated lactational changes, independent of DMBA exposure or dose. Lastly, we demonstrate this strain of virgin female rats has elevated pituitary prolactin immunoreactivity independent of the level of mammary differentiation. We conclude this strain of Charles River Sprague Dawley rats has prolactin-induced pituitary stimulation, and therefore, the window of susceptibility for mammary tumorigenesis is absent.

The renin angiotensin system in the regulation of angiogenesis.

Decades of experimentation on angiotensin and bradykinin have focused on macrovascular systemic effects. However, angiotensin II and bradykinin are both angiogenic agents, highlighting their ability to also effect the microvascular circulation. Not surprisingly, inhibition of angiotensin converting enzyme, which inhibits angiotensin II synthesis and bradykinin degradation, would have different impacts on angiogenesis in vivo dependent upon what factors were present in the system. Several pathological states in which angiogenesis is important, including peripheral ischemia, stroke, retinopathy, and cancer are examined in this review with respect to activity of angiotensin II and bradykinin and the impact of angiotensin converting enzyme inhibition. Although generalizations are not without legitimate criticism, one can think about peripheral ischemia and stroke as being more dependent upon bradykinin signaling and retinopathy and cancer as more dependent upon angiotensin II signaling to drive angiogenesis. Many exceptions are found that are specific to individual animal model systems. Furthermore, cancer systems that have been examined at any depth are few. However, published data on in vitro cultures and animal models present interesting predictions about how the renin angiotensin and bradykinin systems may function in humans. Since angiotensin converting enzyme inhibitors have been widely utilized pharmaceuticals for many years, we are now accumulating epidemiological data that test our predictions. The importance of understanding which agent, angiotensin and/or bradykinin, appears to be the more important regulator of angiogenesis in a given pathology will become increasing evident as more specific angiotensin II and bradykinin receptor blocking drugs make their way into clinical use.

Venous neointimal hyperplasia in polytetrafluoroethylene dialysis grafts.

BACKGROUND: Vascular access dysfunction is the most important cause of morbidity and hospitalization in the hemodialysis population in the United States at a cost of $1 billion per annum. Venous neointimal hyperplasia (VNH) characterized by stenosis and subsequent thrombosis accounts for the overwhelming majority of pathology resulting in polytetrafluoroethylene (PTFE) dialysis graft failure. Despite the magnitude of the problem and the enormity of the cost ($1 billion), there are currently no effective therapies for the prevention or treatment of venous neointimal hyperplasia in PTFE dialysis grafts. METHODS: Tissue samples were collected from the graft-vein anastomosis of stenotic PTFE grafts during surgical revision. Specimens were graded using standard light microscopy and immunohistochemistry for the magnitude of neointimal hyperplasia and for the expression of specific cell types, cytokines, and matrix proteins. RESULTS: VNH was characterized by the (1) presence of smooth muscle cells/myofibroblasts, (2) accumulation of extracellular matrix components, (3) angiogenesis within the neointima and adventitia, and (4) presence of an active macrophage cell layer lining the PTFE graft material. Platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) were expressed by smooth muscle cells/myofibroblasts within the venous neointima, by macrophages lining both sides of the PTFE graft, and by vessels within the neointima and adventitia. CONCLUSIONS: Our results suggest that macrophages, specific cytokines (bFGF, PDGF, and VEGF), and angiogenesis within the neointima and adventitia are likely to contribute to the pathogenesis of VNH in PTFE dialysis grafts. Interventions aimed at these specific mediators and processes may be successful in reducing the very significant human and economic costs of vascular access dysfunction.

DMBA-induced mammary pathologies are angiogenic in vivo and in vitro.

We have previously shown that human pre-invasive diseases of the breast are angiogenic. In addition, normal epithelium from women with coincident or subsequent invasive breast cancer is more vascular than normal epithelium from women with no breast cancer. To develop a model in which to study the regulation of angiogenesis in pre-invasive mammary pathologies, we examined 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary tissues for the presence of neovascularization in pre-invasive histopathologies. These studies included morphometric analysis of tissue vascularity in pre-invasive lesions. In addition, we isolated fresh tumors and histologically normal epithelium (organoids) from DMBA or vehicle-treated control rats to test their ability to induce endothelial cell tubule formation in vitro. Finally, we examined tumors for their ability to produce vascular endothelial cell growth factor. The morphometric studies documented that with epithelial progression, the ability of individual cells to elicit angiogenesis increases. The in vitro studies showed that isolated tumors from these animals stimulate angiogenesis. Furthermore, normal epithelium from DMBA-treated rats is more angiogenic than epithelium from control animals. Finally, DMBA-induced tumors produce vascular endothelial growth factor (VEGF) mRNA, therefore, DMBA-induced mammary tumorigenesis is one model in which to test the dependency of progression on angiogenesis.

Potential role of growth factors and extracellular matrix in wound healing after laryngotracheal reconstruction.

Laryngotracheal reconstruction (LTR) has been used for more than 20 years to treat infants and children with subglottic stenosis. Results after pediatric LTR have been satisfactory; however, approximately 10% of children have recurrent airway narrowing after LTR. The purpose of our study was to determine whether a correlation existed between specific growth factors and extracellular matrix in patients with adequate wound healing capability as compared with patients with poor wound healing capability. Histologic sections from 27 patients who underwent LTR were cut, and immunohistochemical staining was performed for transforming growth factor-beta, platelet-derived growth factor, fibronectin, tenascin, transforming growth factor-alpha, and vascular endothelial growth factor. Results showed that patients with adequate wound healing capability had a positive correlation with vasculature fibronectin, vasculature tenascin, and stromal fibronectin. Patients with poor wound healing capability had a positive correlation with stromal vascular endothelial growth factor.

Expression of Ki-67, tumor suppressor proteins, growth factor, and growth factor receptor in juvenile respiratory papillomatosis: Ki-67 and p53 as predictors of aggressive disease.

The enhanced proliferation of epithelial cells is a typical feature of respiratory papilloma. The mechanism or mechanisms leading to abnormal epithelial proliferation remain unclear. Overexpression of growth factors and their receptors and inactivation of tumor-suppressor proteins are known to cause cell transformation and proliferation. The objectives of this study were to evaluate the expression of these factors in juvenile respiratory papillomas with correlation to cellular proliferation activity, and to determine whether such expression is associated with the clinical course of the disease. The expression of transforming growth factor-alpha, epidermal growth factor receptor, p53 protein, retinoblastoma proteins and Ki-67 was quantified by immunohistochemistry in paraffin-embedded biopsy specimens taken at the initial surgical excision from children in whom respiratory papillomatosis was diagnosed. Clinical information regarding the number of disease sites, tracheobronchial spread, malignant transformation, and frequency of recurrences was reviewed. Thirty-five specimens were suitable for immunohistochemical evaluation. Ki-67 expression was significantly higher in patients with multiple sites of disease and frequent recurrences. High p53 expression was significantly associated with malignant transformation. We concluded that Ki-67 and p53 expression may be predictive of the clinical course in children with respiratory papillomatosis.

Cyclin D1, retinoblastoma, p53, and Her2/neu protein expression in preinvasive breast pathologies: correlation with vascularity.

OBJECTIVES: Preinvasive breast pathologies show a degree of vascularization that correlates with risk of invasion. Recently, numerous oncogenes and tumor suppressor genes have been shown to regulate neovascularization. Therefore, we examined archival tissues of preinvasive breast pathologies by immunohistochemistry for alterations in the expression of four proteins, cyclin D1, retinoblastoma (Rb), p53, and Her2/neu, known to be important in breast tumorgenesis, and correlated these data with tissue vascularity. METHODS: Vascularity was determined by immunologic detection of von Willebrand factor. For carcinoma in situ (CIS) both stromal vascularity (MVD) and vascular cuffing (MCD) were determined. RESULTS: We found that cyclin D1 expression was increased in usual hyperplasia (11% of cases). Atypical hyperplasia, noncomedo CIS and comedo CIS were positive in 43, 49, and 57% of cases, respectively. Changes in Rb and p53 were rare in hyperplasia but occurred in 8 and 10% of CIS, respectively. Her2/neu protein was identified rarely in atypical hyperplasia and in both noncomedo and comedo ductal CIS. Neither Rb nor Her2/neu expression correlated with vascularity. p53 immunoreactivity correlated positively with both MCD and MVD. Cyclin D1 was negatively associated with MVD. CONCLUSION: These data suggest that p53 and cyclin D1 proteins may regulate the microvessel density of preinvasive breast pathologies.

Angiogenic growth factors in preinvasive breast disease.

Recently, we showed that preinvasive breast pathologies, such as usual hyperplasia, atypical hyperplasia, and carcinoma in situ, have an increased vascularity when compared with normal breast tissue (S. C. Heffelfinger et al., Clinical Cancer Res., 2: 1873-1878, 1996). To understand the mechanism of this increased vascularity, we examined by immunohistochemistry each of these pathological lesions for the expression of angiogenic growth factors. These studies showed that normal breast tissue contains numerous angiogenic agents, particularly vascular endothelial cell growth factor and basic fibroblast growth factor. At the transition from normal epithelium to proliferative breast disease, insulin-like growth factor (IGF) II expression was increased, primarily in the stroma and infiltrating leukocytes. However, among proliferative tissues, IGF I decreased with increasing vascularity. Finally, both epithelial vascular endothelial growth factor and epithelial and leukocytic platelet-derived endothelial cell growth factor increased at the transition to carcinoma in situ, whereas stromal and leukocytic basic fibroblast growth factor were elevated only in invasive carcinoma. Therefore, during histological progression there is also a complex progression of angiogenic growth factors. For CIS, two forms of vascularity are found: stromal microvascular density (MVD), and vascularity associated with the epithelial basement membrane (vascular score). There was 35% discordance between these two measurement systems. Among carcinoma in situ cases, decreases in stromal IGF II were associated with increasing vascular scores but not MVD, and increases in platelet-derived endothelial cell growth factor were associated with increasing MVD but not the vascular score. The presence of discordance and differential association with specific angiogenic agents suggests that these two forms of vascularity may be differentially regulated.

Overexpression of transforming growth factor-alpha and epidermal growth factor-receptor in idiopathic pulmonary fibrosis.

BACKGROUND AND AIM: A recent transgenic mouse model overexpressing transforming growth factor alpha (TGF-alpha) led to a phenotype of pulmonary fibrosis. In order to validate this mouse as a model for idiopathic pulmonary fibrosis in humans, we studied the expression of TGF-alpha in lung tissue of patients with idiopathic pulmonary fibrosis compared to control lung tissue. METHODS: Tissue from both groups was obtained from operative specimens and immediately formalin-fixed and paraffin embedded. Contiguous four micron sections were prepared for conventional histochemical staining and staining with antibodies to either TGF-alpha or the epidermal growth factor-receptor (EGF-R). Immunostaining was performed using the Ventana ES automated immunohistochemistry system. Four cell types were examined (vascular endothelium, bronchial epithelium, type 2 pneumocytes, and fibroblasts) and stain activity was scored on a six point scale. RESULTS: Eleven patients with IPF were compared to seven control subjects. TGF-alpha immunoreactivity was significantly higher in the IPF patients than in controls in the vascular endothelium, type 2 pneumocytes, and fibroblasts (P < 0.005). [IPF (4(2-4) Median (Range)) than the controls (0.5(0-2), p < 0.0005).] The differences in EGF-R, one of the receptors for TGF-alpha, between these two patient populations were not as striking. There was a small but significantly greater expression of EGF-R in the bronchial epithelium and type 2 pneumocytes of the IPF patients. CONCLUSIONS: TGF-alpha is overexpressed in patients with IPF, especially in the vascular endothelial cells.

Staurosporine-induced versus spontaneous squamous metaplasia in pre- and postmenopausal breast tissue.

Breast cancers from pre- vs. postmenopausal women display unique characteristics that may be related to differences in epithelial differentiation between these two populations. In addition to lobular development, lactational changes, and involution, breast epithelium can undergo metaplastic alterations, often in association with carcinoma. Because protein kinase C (PKC) regulates differentiation and proliferation in many cell types, we asked whether modulation of PKC activity could define biochemical differences in breast epithelium from pre- vs. postmenopausal women. Organ cultures of normal human breast were treated with PKC agonists and antagonists. Epithelial differentiation was evaluated based on morphologic criteria and the expression of cell-type specific proteins. Staurosporine, a nonspecific but extremely potent inhibitor of PKC, induced squamous metaplasia in eight of eight cases within 2 weeks of treatment. Other inhibitors of PKC, such as calphostin C and tamoxifen, had no effect on epithelial differentiation. Long-term treatment with phorbol esters also did not induce squamous metaplasia. However, stimulation of cAMP levels by forskolin and isobutyl-methyl-xanthene (IMX) rapidly induced squamous metaplasia, as has been previously reported. Surprisingly, squamous metaplasia occurred in 10 of 12 cultures derived from postmenopausal women in the absence of exogenous agents. Untreated cultures derived from premenopausal women never developed this type of epithelium (0 of 11). Therefore, breast epithelium from pre- and postmenopausal women responded differently to in vitro culture. Forskolin/IMX or staurosporine can reproduce these conditions, acting independent of menopausal status. Because staurosporine's action was unique among PKC inhibitors, staurosporine may induce squamous metaplasia of breast epithelium by a PKC-independent mechanism.

Plasma membrane phosphotyrosine, Her2-NEU, and epidermal growth factor receptor in human breast cancer. A comparative study.

Experimental therapeutic regimens for breast cancer include strategies to block the activity of specific oncogenes. Because oncogenesis is a multistep process, specific oncogenes may drive tumor production at one stage yet not function in another. Since the effectiveness of therapy targeted against oncogenes depends on their function in the tumor, correlation of oncogene function to specific stages of tumor development has therapeutic implications. Among the oncogenes known to be important in breast cancer production are two cell surface growth factor receptors, epidermal growth factor receptor (EGFR) and Her2-NEU (NEU). These proteins are receptor tyrosine kinases that autophosphorylate specific tyrosine residues on activation. The oncogenic potential of these receptors depends on this autophosphorylation. We examined 86 primary formalin-fixed, paraffin-embedded breast tumors for overexpression of EGFR and NEU and correlated our findings with the presence of cell surface phosphotyrosine as an indicator of tyrosine kinase activity at the plasma membrane. Our data indicate that only 34% of tumors that overexpress EGFR or NEU show plasma membrane phosphotyrosine, indicating that in the majority of these tumors, the overexpressed oncogene may not be active at this stage.

Vascularity of proliferative breast disease and carcinoma in situ correlates with histological features.

The level of vascularity within an invasive breast carcinoma is a predictor of metastatic potential and survival. However, little is known about the vascular potential and prognostic value of angiogenesis in preinvasive breast pathology. Women with proliferative breast disease or carcinoma in situ are at increased risk of developing invasive breast cancer. This relative risk increases in correlation with defined histopathological features. We asked whether these early proliferative lesions and carcinoma in situ were capable of inducing a vascular supply. Vascularity in preinvasive archival paraffin-embedded breast tissue from 90 patients was quantified by immunohistochemical identification of vessels using anti-von Willebrand factor. Vascular scores were analyzed with respect to histopathological diagnosis, age at diagnosis, and presence of coincident invasive disease. These data indicate that: (a) the vascularity of histopathologically normal epithelium is greater in breasts containing invasive disease than in breasts lacking invasive disease; (b) simple proliferative breast disease induces a vascular supply greater than that of normal breast epithelium; and (c) vascularity increases in proportion to epithelial lesion progression and relative risk of invasion. These studies indicate that the vascularity of preinvasive breast pathology may be a clinically useful predictor of invasive breast cancer.

RNA expression of complement regulatory proteins in human brain tumors.

The expression of complement regulatory proteins (CRP) on the surface of neoplastic cells has been proposed as a mechanism by which these cells evade immune surveillance. We have examined the RNA expression of the genes that encode 5 kinds of CRP in various human brain tumors to determine whether CRP expression might play a role in the malignant progression of these tumors. The benign and atypical meningiomas, and the astrocytomas showed high expression of SP-40,40, low expression of CD59, and barely detectable expression of CD46, CD55 and S-protein. The benign and atypical menigiomas showed significantly greater expression of SP-40,40 at the RNA level when compared to malignant meningiomas. This study describes the mRNA expression of meningiomas, astrocytomas, tumor cell lines and normal human tissues.

The molecular biology of esophageal and gastric cancer and their precursors: oncogenes, tumor suppressor genes, and growth factors.

The evolution of sequential histological changes from normal cells through invasive cancer affords the cancer biologist the opportunity to identify separate molecular steps involved in cancer progression. As one studies the development of human carcinoma, it becomes apparent that multiple genetic alterations affecting both cellular proto-oncogenes and tumor suppressor genes are involved during the development and progression of both esophageal and gastric cancers. The different histological forms of both esophageal and gastric carcinomas as well as their differing etiologies result in the possibility that a spectrum of genetic changes is involved in different tumor types. p53 abnormalities occur frequently in tumors arising in both organs, and in both sites p53 abnormalities can be observed in precancerous lesions as well as in overt cancer. Subsequent abnormalities affecting other genes (eg, epithelial growth factor receptors [EGFRs]) potentially enhance the growth potential of tumors. This review focuses on abnormalities of oncogenes, tumor suppressor genes, and growth factors commonly found in cancers of the esophagus and stomach.

SK HEP-1: a human cell line of endothelial origin.

SK-HEP-1 is an immortal, human cell line derived from the ascitic fluid of a patient with adenocarcinoma of the liver. We have determined that these cells are of endothelial origin. Despite the location of the tumor from which SK HEP-1 was derived, the cell line does not have properties of hepatocytes. Northern blot analysis of total cellular RNA shows no messenger RNA for the hepatic-specific proteins albumin, alpha-fibrinogen, or gamma-fibrinogen. Endothelial characteristics are seen by transmission electron microscopy. These features include numerous pinocytotic vesicles, electron dense granules consistent with Weibel-Palade bodies, and abundant intermediate filaments, identified immunocytochemically as vimentin. Cultures grown on plastic dishes grow in bundles of polygonal to spindle-shaped cells. Proteins characteristic for endothelial cells are identified by immunocytochemistry. Addition of basement membrane material (Matrigel) or type I collagen to the cultures induces these cells to organize into a tubular network.

Leukocyte adhesion deficiency: clinical and postmortem observations.

The clinical and autopsy findings in a patient with the severe form of leukocyte adhesion deficiency are presented. An 18-month-old Hispanic female had a history of delayed umbilical cord separation, recurrent necrotizing skin lesions, and gingivitis. Her neutrophils were found to lack detectable CD11/CD18 adhesion glycoproteins and were deficient in adhesion-dependent functions. She succumbed to necrotizing enterocolitis, peritonitis, and pneumonia following sudden cardiorespiratory collapse. Postmortem examination revealed multiple regions of mucosal ulceration and bacterial and fungal overgrowth with complete lack of an acute inflammatory response. Impaired neutrophil emigration from blood vessels into injured tissue appears to have been the basis of this patient's disease. Some of the many foci of bronchopneumonia, in contrast, contained numerous neutrophils. Lymphoid tissue, including the thymus, was severely depleted of lymphocytes. These findings support the concepts that neutrophils can emigrate in response to certain stimuli via CD18-independent mechanisms and that severe deficiency of CD18 is associated with compromised function of lymphocytes in vivo.

Xanthomatous bronchiolitis obliterans with cholesterol pneumonia.

Reported is an instance of bronchiolitis obliterans due to the accumulation of lipid-laden cells within bronchiolar walls and lumina. The bronchiolar lining had extensive squamous and goblet cell metaplasia with focal ulceration. Scattered foci of cholesterol pneumonia, composed of acicular crystals with an associated foamy lipid material and type II alveolar epithelial cell hyperplasia, were present. The patient had no history related to this unusual pulmonary disease, although preoperatively her serum cholesterol level was mildly elevated. She was treated with pulmonary and cardiac allotransplantation.

Absence of branched chain acyl-transferase as a cause of maple syrup urine disease.

Decreased function of human mitochondrial branched chain alpha-ketoacid dehydrogenase complex results in branched chain ketoacidemia or maple syrup urine disease. Activity of this multienzyme complex varies from 0 to approximately 15% of wild type branched chain alpha-ketoacid dehydrogenase complex activity within the population of homozygous affected individuals. We used the technique of Western Blotting with antibodies against purified bovine liver branched chain alpha-ketoacid dehydrogenase complex to screen mitochondrial proteins from cultured human fibroblasts for immunocrossreactive proteins. This method probes the physical structure of the proteins forming this multienzyme complex. One patient with branched chain ketoacidemia lacked an immunoreactive transacylase protein. This protein catalyzes the transfer of the branched chain acyl group from the decarboxylase to reduced coenzyme A. Kinetic analysis of the enzyme activity in cell lysates from this patient confirmed that the complex would not utilize coenzyme A. Thus, we have defined a structural basis for an impaired multienzyme complex of mitochondria in man.

Direct physical evidence for stabilization of branched-chain alpha-ketoacid dehydrogenase by thiamin pyrophosphate.

Branched-chain alpha-ketoacid dehydrogenase is a multienzyme complex composed of four subunits. The 46,500-dalton protein is a subunit of the decarboxylase component, which is selectively digested by chymotrypsin. Two peptides of apparent mol. wts. of 36,000 and 15,000 result with loss of enzyme activity. When the complex is saturated with thiamin pyrophosphate and ketoacid substrate, digestion by chymotrypsin does not occur. These data provide direct physical evidence for the stabilization of the complex by the presence of the vitamin B1-derived cofactor.