Tova Chajek-Shaul

A recessive mutation in desmoplakin causes arrhythmogenic right ventricular dysplasia, skin disorder, and woolly hair.

OBJECTIVESnThe goal of this study was to analyze the genetic disorder of a family with cardiomyopathy, skin disorder, and woolly hair.nnnBACKGROUNDnArrhythmogenic right ventricular dysplasia (ARVD) is a heart muscle disorder causing arrhythmia and sudden cardiac death. We report a patient with familial autosomal recessive ARVD, woolly hair, and a pemphigous-like skin disorder with a new mutation in the desmoplakin gene.nnnMETHODSnGenomic deoxyribonucleic acid was extracted from the patients blood and 12 first- and second-degree family members, and was amplified by polymerase chain reaction. Linkage analysis with polymorphic microsatellites was performed for 11 genes that code for structural desmosomal proteins. The genetic locus of the disease in this family was mapped to the chromosomal region 6p24 that contains the desmoplakin gene. Exons of the desmoplakin gene were analyzed by single-strand conformational polymorphism and direct sequencing. Confirmation of the mutation was carried out by restriction enzyme analysis.nnnRESULTSnWe identified in the patient a homozygous missense mutation in exon 24 of the desmoplakin gene, leading to a Gly2375Arg substitution in the C-terminal of the protein where the binding site to intermediate filaments is located. Eight of 12 family members without hair or skin abnormalities were heterozygous for this mutation. The remaining 4, as well as 90 unrelated healthy control individuals of the same ethnic origin, were homozygous for the normal allele.nnnCONCLUSIONSnWe have described a new mutation in the desmoplakin gene that causes familial ARVD. These findings suggest that desmosomal proteins play an important role in the integrity and function of the myocardium. Dysfunction of these proteins can lead to the development of cardiomyopathies and arrhythmias.

Transgenic expression of mammalian heparanase uncovers physiological functions of heparan sulfate in tissue morphogenesis, vascularization, and feeding behavior

We have generated homozygous trans¬genic mice (hpa‐tg) overexpressing human hepara¬nase (endo‐β‐D‐glucuronidase) in all tissues and char¬acterized the involvement of the enzyme in tissue morphogenesis, vascularization, and energy metabo¬lism. Biochemical analysis of heparan sulfate (HS) isolated from newborn mice and adult tissues re¬vealed a profound decrease in the size of HS chains derived from hpa‐tg vs. control mice. Despite this, the mice appeared normal, were fertile, and exhib¬ited a normal life span. A significant increase in the number of implanted embryos was noted in the hpa‐tg vs. control mice. Overexpression of heparanase resulted in increased levels of urinary protein and creatinine, suggesting an effect on kidney func¬tion, reflected also by electron microscopy examina¬tion of the kidney tissue. The hpa‐tg mice exhibited a reduced food consumption and body weight com¬pared with control mice. The effect of heparanase on tissue remodeling and morphogenesis was best dem¬onstrated by the phenotype of the hpa‐tg mammary glands, showing excess branching and widening of ducts associated with enhanced neovascularization and disruption of the epithelial basement membrane. The hpa‐tg mice exhibited an accelerated rate of hair growth, correlated with high expression of heparanase in hair follicle keratinocytes and increased vascularization. Altogether, characterization of the hpa‐tg mice emphasizes the involvement of heparanase and HS in processes such as embryonic implan¬tation, food consumption, tissue remodeling, and vascularization.—Zcharia, E., Metzger, S., ChajekShaul, T., Aingorn, H., Elkin, M., Friedmann, Y., Weinstein, T., Li, J.‐P., Lindahl, U., Vlodavsky, I. Transgenic expression of mammalian heparanase uncovers physiological functions of heparan sulfate in tissue morphogenesis, vascularization, and feeding behavior. FASEB J. 18, 252–263 (2004)

Mechanism of the hypertriglyceridemia induced by tumor necrosis factor administration to rats

4 h after intravenous injection of recombinant HuTNF-alpha to fed rats, an increase in heart, diaphragm, and plasma lipoprotein lipase activity was observed. At the same time, a 40-60% decrease in enzymic activity in epididymal fat pad and kidney and 40% decrease in hepatic lipase activity in liver had occurred. Similar results were obtained 20 h after injection of recombinant HuTNF-alpha into fasted rats. Pretreatment with Indomethacin did not affect the changes in tissue lipoprotein lipase activity observed following recombinant HuTNF-alpha administration. Serum triacylglycerol concentration increased by 2- and 6-fold; 4 and 20 h after recombinant HuTNF-alpha administration. Disappearance of 14C-labeled triacylglycerol from the circulation after injection of small chylomicrons, biosynthetically labeled in their triacylglycerol and cholesterol moieties, was lower in TNF-treated than in control rats. However, the clearance rate of triacylglycerol was the same or even higher in recombinant HuTNF-alpha treated rats (assuming that 14C-labeled chylomicron triacylglycerol represents the serum triacylglycerol pool). The livers of recombinant HuTNF-alpha-treated rats and controls contained similar amounts of 14C-labeled lipids, but less [3H]cholesterol, suggesting that in recombinant HuTNF-alpha-treated rats, the liver took up chylomicron remnant particles enriched with triacylglycerol. Separation of the d less than 1.04 g/ml fraction of serum obtained from control and recombinant HuTNF-alpha treated rats by zonal ultracentrifugation revealed that in recombinant HuTNF-alpha-treated rats the lipoprotein particles were less lipolyzed than in controls. The secretion rate of [3H]triacylglycerol into the serum was determined 90 min after injection of [3H]palmitate albumin complex and Triton WR 1339. In recombinant HuTNF-alpha-treated rats, the secretion of [3H]triacylglycerol into plasma was 48% higher than in controls. It is suggested that the increase in lipoprotein lipase activity of heart and diaphragm resulted from an indirect effect of TNF. It is concluded that the increase in serum triacylglycerol in the recombinant HuTNF-alpha-treated rats is due mainly to an increased secretion of triacylglycerol by the liver. Impaired lipolysis, probably due to a fall in hepatic lipase could also contribute to the rise in plasma triacylglycerol.

Low dose warfarin treatment for calcinosis in patients with systemic sclerosis

Objective: To evaluate the effect of low doses of warfarin in patients with systemic sclerosis with disseminated subcutaneous calcinosis. Methods: Three patients with disseminated subcutaneous calcinosis were treated with low doses of warfarin for 1 year. Subcutaneous calcinotic lesions, coagulation blood parameters, and the tendency for bleeding were followed up during the year. Results: Two of the patients, who had newly diagnosed, diffuse, and relatively small calcinotic lesions, responded to warfarin treatment, with complete resolution of the calcinosis. The other patient, with larger and longer standing calcinotic lesions, did not respond to warfarin treatment. None of the three patients showed a prolongation of prothrombin time or partial thromboplastin time, nor did any have an increased tendency for bleeding. Conclusions: Low dose warfarin may serve as an effective treatment for calcinosis in a selected group of patients who have small and relatively new onset calcinosis. This treatment does not prolong the coagulation of blood and there is no increased tendency for bleeding.

Molecular Properties and Involvement of Heparanase in Cancer Progression and Mammary Gland Morphogenesis

Tumor spread involves degradation of various components of the extracellular matrix and blood vessel wall. Among these is heparan sulfate proteoglycan, which plays a key role in the self-assembly, insolubility and barrier properties of basement membranes and extracellular matrices. Expression of an endoglycosidase (heparanase) which degrades heparan sulfate correlates with the metastatic potential of tumor cells, and treatment with heparanase inhibitors markedly reduces the incidence of metastasis in experimental animals. Heparin-binding angiogenic proteins are stored as a complex with heparan sulfate in the microenvironment of tumors. These proteins are released and can induce new capillary growth when heparan sulfate is degraded by heparanase. Here, we describe the molecular properties, expression and involvement in tumor progression of a human heparanase. The enzyme is synthesized as a latent ∼65 kDa protein that is processed at the N-terminus into a highly active ∼50 kDa form. The heparanase mRNA and protein are preferentially expressed in metastatic human cell lines and in tumor biopsy specimens, including breast carcinoma. Overexpression of the heparanase cDNA in low-metastatic tumor cells conferred a high metastatic potential in experimental animals, resulting in an increased rate of mortality. The heparanase enzyme also released ECM-resident bFGF in vitro, and its overexpression elicited an angiogenic response in vivo. Heparanase may thus facilitate both tumor cell invasion and neovascularization, two critical steps in tumor progression. Mammary glands of transgenic mice overexpressing the heparanase enzyme exhibit precocious branching of ducts and alveolar development, suggesting that the enzyme promotes normal morphogenesis and possibly pre-malignant changes in the mammary gland.

Molecular properties and involvement of heparanase in cancer progression and normal development.

Heparan sulfate proteoglycans (HSPGs) play a key role in the self-assembly, insolubility and barrier properties of basement membranes and extracellular matrices. Hence, cleavage of heparan sulfate (HS) affects the integrity and functional state of tissues and thereby fundamental normal and pathological phenomena involving cell migration and response to changes in the extracellular microenvironment. Here, we describe the molecular properties, expression and function of a human heparanase, degrading HS at specific intrachain sites. The enzyme is synthesized as a latent approximately 65 kDa protein that is processed at the N-terminus into a highly active approximately 50 kDa form. The heparanase mRNA and protein are preferentially expressed in metastatic cell lines and human tumor tissues. Overexpression of the heparanase cDNA in low-metastatic tumor cells conferred a high metastatic potential in experimental animals, resulting in an increased rate of mortality. The heparanase enzyme also releases ECM-resident angiogenic factors in vitro and its overexpression induces an angiogenic response in vivo. Heparanase may thus facilitate both tumor cell invasion and neovascularization, both critical steps in cancer progression. The enzyme is also involved in cell migration associated with inflammation and autoimmunity. The unexpected identification of a single predominant functional heparanase suggests that the enzyme is a promising target for drug development. In fact, treatment with heparanase inhibitors markedly reduces tumor growth, metastasis and autoimmune disorders in animal models. Studies are underway to elucidate the involvement of heparanase in normal processes such as implantation, embryonic development, morphogenesis, tissue repair, inflammation and HSPG turnover. Heparanase is the first functional mammalian HS-degrading enzyme that has been cloned, expressed and characterized. This may lead to identification and cloning of other glycosaminoglycan degrading enzymes, toward a better understanding of their involvement and significance in normal and pathological processes.

Heparanase is expressed in osteoblastic cells and stimulates bone formation and bone mass

Heparan sulfate proteoglycans (HSPGs) are ubiquitous macromolecules. In bone, they are associated with cell surfaces and the extracellular matrix (ECM). The heparan sulfate (HS) chains of HSPGs bind a multitude of bioactive molecules, thereby controlling normal and pathologic processes. The HS‐degrading endoglycosidase, heparanase, has been implicated in processes such as inflammation, vascularization associated with wound healing and malignancies, and cancer metastasis. Here we show progressive mRNA expression of the hpa gene (encoding heparanase) in murine bone marrow stromal cells undergoing osteoblastic (bone forming) differentiation and in primary calvarial osteoblasts. Bone marrow stromal cells derived from transgenic mice expressing recombinant human heparanase (rh‐heparanase) and MC3T3 E1 osteoblastic cells exposed to soluble rh‐heparanase spontaneously undergo osteogenic differentiation. In addition, the transgenic bone marrow stromal cells degrade HS chains. In wild‐type (WT) and hpa‐transgenic (hpa‐tg) mice, heparanase is weakly expressed throughout the bone marrow with a substantial increase in osteoblasts and osteocytes, especially in the hpa‐tg mice. Heparanase expression was absent in osteoclasts. Micro‐computed tomographic and histomorphometric skeletal analyses in male and female hpa‐tg versus WT mice show markedly increased trabecular bone mass, cortical thickness, and bone formation rate, but no difference in osteoclast number. Collectively, our data suggest that proteoglycans tonically suppress osteoblast function and that this inhibition is alleviated by HS degradation with heparanase. J. Cell. Physiol.

Association of the MIC-A gene and HLA-B51 with Behçet's disease in Arabs and non-Ashkenazi Jews in Israel

Background: Behçets disease is known to be strongly associated with HLA-B51 in many different ethnic groups. Recently, it was suggested that MIC-A (major histocompatibility complex class I related gene A) is the pathogenic gene after strong association was found between the MIC-A A6 allele of the transmembrane region and the disease in Japanese and Greek patients, although in Greek patients this association was found to be due to linkage disequilibrium with HLA-B51. Objectives: To investigate microsatellite polymorphism in Arab and non-Ashkenazi Jewish (NAJ) patients in Israel, to determine whether this association occurs in these groups with Behçets disease, and elucidate the associated HLA allele of the disease. Methods: Forty four Israeli patients with Behçets disease, including 20 Arabs and 24 NAJ, and 130 ethnically matched healthy controls were examined for MIC-A microsatellite polymorphism of the transmembrane region using polymerase chain reaction, autoradiography, and sequence analysis. Results: The MIC-A A6 allele was significantly more frequent in the Arab patient group (19/20 (95%)) than in healthy Arab controls (25/42 (60%)) (pcorr=0.015, OR=12.92), but not in the NAJ patients (16/24 (67%)) compared with NAJ healthy controls (48 /88 (55%)) (pcorr=1.02, OR=1.667). In stratification analysis of the Arab subgroup, on the confounding effect of MIC-A A6 on HLA-B51 association and vice versa, Behçets disease was distinctly associated only with HLA-B51. Conclusions: These results imply strong association between the MIC-A A6 allele and the disease in Israeli Arabs, but not in Israeli NAJ patients. The stratification analysis indicates that this association results secondarily from a strong linkage disequilibrium with HLA-B51, and the real disease susceptibility gene which plays a part in the development of Behçets disease is most probably the HLA-B51 allele itself.

Brain Natriuretic Peptide Levels Predict Perioperative Events in Cardiac Patients Undergoing Noncardiac Surgery: A Prospective Study

Objectives: Brain natriuretic peptide (BNP) levels correlate with prognosis in patients with cardiac disease and may be useful in the risk stratification of cardiac patients undergoing noncardiac surgery (NCS). The objective of this study was to examine whether BNP levels predict perioperative events in cardiac patients undergoing NCS. Methods: Patients undergoing NCS with at least 1 of the following criteria were included: a clinical history of congestive heart failure (CHF), ejection fraction <40%, or severe aortic stenosis. All patients underwent echocardiography and measurement of BNP performed using the ADVIA-Centaur BNP assay (Bayer HealthCare). Clinical endpoints were death, myocardial infarction or pulmonary congestion requiring intravenous diuretics at 30 days of follow-up. Results: Forty-four patients were entered into the study; 15 patients (34%) developed cardiac postoperative complications. The mean BNP level was 1,366 ± 1,420 pg/ml in patients with events and 167 ± 194 pg/ml in patients without events, indicating a highly significant difference (p < 0.001). The ROC area under the curve was 0.91 (95% CI 0.83–0.99) with an optimal cutoff of >165 pg/ml (100% sensitivity, 70% specificity). Conclusions: BNP levels may predict perioperative complications in cardiac patients undergoing NCS, and the measurement of BNP should be considered to assess the preoperative cardiac risk.

Uptake of chylomicron [3H]cholesteryl linoleyl ether by mesenchymal rat heart cell cultures

Abstract Rat heart cell cultures were used to study uptake of chylomicron cholesteryl ester, using a nondegradable analog, cholesteryl linoleyl ether. Rat mesenteric duct chylomicrons were labeled biosynthetically with palmitate and with [ 3 H]cholesteryl linoleyl ether or [ 14 C]cholesteryl ester by transfer from high-density lipoprotein. Rat heart cell cultures incubated with chylomicrons labeled with [ 3 H]cholesteryl linolpeyl ether and [ 14 C]cholesteryl ester took up both lipids to the same extent, and the intracellular degradation of the labeled cholesteryl ester was inhibited by chloroquine. The uptake of [ 3 H]cholesteryl linoleyl ether exceeded that of 125 I-labeled chylomicron protein by an order of magnitude, indicating that these may be nonrelated phenomena. The extent of uptake of the labeled cholesteryl linoleyl ether was correlated with the activity of endogenous lipoprotein lipase of the heart cell cultures. However, the uptake of the labeled cholesteryl ether from intact chylomicrons and from chylomicron remnants, prepared by a membrane-supported lipoprotein lipase in three different systems, was the same. These findings suggest that lipoprotein lipase may play a role in cellular uptake of chylomicron cholesteryl ester independently of hydrolysis of triacylglycerol. This hypothesis was further tested and the results are presented in the accompanying paper (Friedman, G., Chajek-Shaul, T., Stein, O., Olivecrona, T. and Stein, Y. (1981) Biochim. Biophys. Acta 666, 156–164).