Angel Panizo

Serum Markers of Collagen Type I Metabolism in Spontaneously Hypertensive Rats Relation to Myocardial Fibrosis

BACKGROUND The assay of serum peptides of extracellular collagen synthesis and degradation could provide an indirect estimate of the rate of fibrillar turnover. This study was designed to investigate whether serum peptides of collagen type I synthesis and degradation are altered in spontaneously hypertensive rats (SHR) with left ventricular hypertrophy and whether these serum collagen-derived peptides are related to myocardial fibrosis. METHODS AND RESULTS We measured serum levels of carboxyterminal propeptide of procollagen type I (PIP) as a marker of collagen I synthesis and serum levels of the pyridinoline crosslinked telopeptide domain of collagen type I (CITP) as a marker of fibrillar collagen I degradation in ten 36-week-old normotensive Wistar-Kyoto (WKY) rats, ten 36-week-old SHR, and ten 16-week-old SHR treated with the angiotensin-converting enzyme inhibitor quinapril (10 mg /kg body wt per day, orally) for 20 weeks. PIP and CITP were determined by specific radioimmunoassays. Histomorphometric and immunohistochemical studies of the left ventricle were performed in all rats. In untreated SHR compared with WKY rats, we found a more extensive interstitial and perivascular fibrosis, an increased (P<.01) collagen volume fraction, a more marked deposition of collagen type I, an increased (P<.01) serum concentration of PIP, and a similar serum concentration of CITP. In quinapril-treated SHR compared with untreated SHR, we found an absence of left ventricular hypertrophy, a marked decrease of fibrosis, a lower (P<.01) collagen volume fraction, a diminished deposition of collagen type I, a decreased (P<.01) concentration of PIP, and a similar concentration of CITP. A direct correlation was found between the collagen volume fraction and serum PIP (r=.753, P<.05) in untreated SHR. CONCLUSIONS These results suggest that tissue metabolism of collagen type I is abnormal in SHR and can be normalized by treatment with quinapril. On the basis of our findings, we propose that serum PIP may be a marker of collagen type I-dependent myocardial fibrosis in rats with genetic hypertension.

Association of EWS-FLI1 Type 1 Fusion with Lower Proliferative Rate in Ewing's Sarcoma

The Ewings sarcoma (ES) family of tumors, including peripheral neuroectodermal tumor (PNET), is defined genetically by specific chromosomal translocations resulting in fusion of the EWS gene with a member of the ETS family of transcription factors, either FLI1 (90-95%) or ERG (5-10%). A second level of molecular genetic heterogeneity stems from the variation in the location of the translocation breakpoints, resulting in the inclusion of different combinations of exons from EWS and FLI1 (or ERG) in the fusion products. The most common type of EWS-FLI1 fusion transcript, type 1, is associated with a favorable prognosis and appears to encode a functionally weaker transactivator, compared to other fusion types. We sought to determine whether the observed covariation of structure, function, and clinical course correlates with tumor cell kinetic parameters such as proliferative rate and apoptosis, and with expression of the receptor for insulin-like growth factor I (IGF-1R). In a group of 86 ES/PNET with defined EWS-ETS fusions (45 EWS-FLI1 type 1, 27 EWS-FLI1 non-type 1, 14 EWS-ERG), we assessed proliferation rate by immunostaining for Ki-67 using MIB1 antibody (n = 85), apoptosis by TUNEL assay (n = 66), and IGF-1R expression by immunostaining with antibody 1H7 (n = 78). Ki-67 proliferative index was lower in tumors with EWS-FLI1 type 1 than those with non-type 1 EWS-FLI1, whether analyzed as a continuous (P = 0.049) or categorical (P = 0.047) variable. Logistic regression analysis suggests that this association was secondary to the association of type 1 EWS-FLI1 and lower IGF-1R expression (P = 0.04). Comparing EWS-FLI1 to EWS-ERG cases, Ki-67 proliferative index was higher in the latter (P = 0.01, Mann-Whitney test; P = 0.02, Fishers exact test), but there was no significant difference in IGF-1R. TUNEL results showed no significant differences between groups. Our results suggest that clinical and functional differences between alternative forms of EWS-FLI1 are paralleled by differences in proliferative rate, possibly mediated by differential regulation of the IGF-1R pathway.

Imatinib Inhibits Proliferation of Ewing Tumor Cells Mediated by the Stem Cell Factor/KIT Receptor Pathway, and Sensitizes Cells to Vincristine and Doxorubicin-Induced Apoptosis

Purpose and Experimental Design: The stem cell factor/KIT receptor loop may represent a novel target for molecular-based therapies of Ewing tumor. We analyzed the in vitro impact of KIT blockade by imatinib in Ewing tumor cell lines. Results: KIT expression was detected in 4 of 4 Ewing tumor cell lines and in 49 of 110 patient samples (44.5%) by immunohistochemistry and/or Western blot analysis. KIT expression was stronger in Ewing tumors showing EWS-FLI1 nontype 1 fusions. Despite absence of c-kit mutations, constitutive and ligand-inducible phosphorylation of KIT was found in all tumor cell lines, indicating an active receptor. Treatment with KIT tyrosine kinase inhibitor imatinib (0.5–20 μm) induced down-regulation of KIT phosphorylation and dose response inhibition of cell proliferation (IC50, 12–15 μm). However, imatinib administered alone at doses close to IC50 for growth inhibition (10 μm) did not induce a significant increase in apoptosis. We then analyzed if blockade of KIT loop through imatinib (10 μm) was able to increase the antitumor in vitro effect of doxorubicin (DXR) and vincristine (VCR), drugs usually used in Ewing tumor treatment. Addition of imatinib decreased in 15–20 and 15–36% of the proliferative rate of Ewing tumor cells exposed to DXR and VCR, respectively, and increased in 15 and 30% of the apoptotic rate of Ewing tumor cells exposed to the same drugs. Conclusions: Inhibition of Ewing tumor cell proliferation by imatinib is mediated through blockade of KIT receptor signaling. Inhibition of KIT increases sensitivity of these cells to DXR and VCR. This study supports a potential role for imatinib in the treatment of Ewing tumor.

Are mast cells involved in hypertensive heart disease

Objective: To evaluate the potential relationship of mast cells with myocardial fibrosis in the cardiac ventricles of spontaneously hypertensive rats (SHR). Design: Experiments were performed on hearts from 36-week-old SHR with established left ventricular hypertrophy (n=12) and from 36-week-old normotensive Wistar-Kyoto (WKY) rats (n=12). Furthermore, to evaluate whether antihypertensive treatment with the angiotensin converting enzyme inhibitor quinapril interferes with the potential relationship between mast cells and fibrosis in SHR, we treated 16-week-old SHR (n=12) with oral quinapril (10 mg/kg body weight per day) for 20 weeks. Methods: Mast cells were counted in 25 high-power fields. Toluidine blue-stained sections and avidin staining were used to detect mast cells. The extent of myocardial fibrosis was analysed in samples stained with Massons trichrome. The amount of collagen was evaluated morphometrically, using an automatic image analyser, and biochemically, using myocardial hydroxyproline concentration. Results: In the left ventricle of untreated SHR compared with age- and sex-matched normotensive WKY rats we found more extensive interstitial and perivascular fibrosis, an increased collagen volume fraction, an increased hydroxyproline concentration and an increased number of mast cells. Similar but less intense abnormalities were observed in the right ventricles of untreated SHR compared with the left ventricles of the same rats. In the left ventricles of quinapril-treated SHR compared with those of untreated SHR we found a marked decrease in fibrosis, a lower collagen volume fraction, a lower hydroxyproline concentration and fewer mast cells. Treatment with quinapril was also accompanied by normalization in the myocardial structure of the right ventricles of SHR. A positive correlation was found between the density of mast cells and the collagen volume fraction in the left ventricles of all of the rats. Conclusions: The present findings suggest that mast cells can play a part in the development of the myocardial fibrosis that occurs in the cardiac ventricles with hypertensive cardiac hypertrophy. In addition, the present results suggest that the ability of quinapril to interfere with mast cells might be involved in its cardioreparative properties.

Is the Regulation of Apoptosis Altered in Smooth Muscle Cells of Adult Spontaneously Hypertensive Rats

Whereas the protein product of the Bcl-2 gene inhibits apoptosis, the protein product of the Bax gene acts as a promoter of apoptosis. To gain insight into the regulation of apoptosis in vascular smooth muscle cells in arterial hypertension, we investigated the expression of the proteins Bcl-2 and Bax in small intramyocardial arteries of 36-week-old normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). In addition, 16-week-old SHR were treated for 20 weeks with the angiotensin-converting enzyme inhibitor quinapril and killed at 36 weeks of age. We measured the percentages of smooth muscle cells expressing these proteins using monoclonal antibodies and the avidin-biotin immunoperoxidase method. Compared with WKY, untreated SHR exhibited increased (P<.001) Bcl-2 expression and similar Bax expression. Values of Bcl-2 measured in quinapril-treated SHR were significantly lower than values measured in untreated SHR and similar to values measured in WKY. Quinapril-treated SHR showed higher (P<.001) Bax expression than WKY and untreated SHR. Bcl-2 expression was directly correlated with systolic pressure. Inverse correlations were found between the expression of Bax and the activities of both cardiac and circulating angiotensin-converting enzyme. These findings suggest that smooth muscle cell apoptosis might be inhibited in small arteries of adult SHR as a consequence of an excess of the protein Bcl-2. In addition, our results suggest that chronic angiotensin-converting enzyme inhibition might restore the susceptibility to apoptosis in these cells through stimulation of the protein Bax.

Large cell carcinoma of the lung: an endangered species?

This study aims to evaluate large cell carcinomas (LCC) of the lung with a panel of immunohistochemical markers in an attempt to identify tumors belonging to other categories. We analyzed a tissue microarray platform of 101 LCC with a panel of 31 monoclonal antibodies. The tumors were 82 (81.3%) classic LCC, 7 (6.9%) neuroendocrine LCC, 6 (5.9%) lymphoepithelioma-like LCC, 3 (2.9%) basaloid LCC, 2 (2%) clear cell LCC, and 1 (1%) LCC with rhabdoid phenotype. Characteristic classic LCC immunophenotype was loss of staining with CK5/6, CK14 positive in most squamous cell carcinoma (SCC), lack of MOC 31 positive in most adenocarcinomas, and positive immunoreactivity to EGFR, PDGFR-α and c-kit. 27 of 82 classic LCC (32.9%) were re-classified as adenocarcinomas, because they coexpressed TTF-1, CK7, and CK19, and were negative for p63. 31 (37.8%) of 82 classic LCC were reclassified as poorly differentiated SCC, based on their immunoreactivity with 34βE12, p63, thrombomodulin, and CD44v6. 16 (19.5%) of 82 classic LCC correspond to undifferentiated adenosquamous carcinomas, since they displayed conflicting immunostaining for markers of both SCC and adenocarcinomas. The use of 7 immunohistochemical markers, consisting of TTF-1, CK7, CK19, p63, 34βE12, thrombomodulin, and CD44v6, markedly reduces dramatically to less than 10%, the number of classic LCC by readily identifying cases of poorly differentiated SCCs, adenosquamous carcinoma and adenocarcinomas.

Quinapril decreases myocardial accumulation of extracellular matrix components in spontaneously hypertensive rats

In genetic and acquired hypertension, a structural remodeling of the nonmyocyte compartment of myocardium, including the accumulation of fibrillar collagen and other components of the extracellular matrix (ECM) within the interstitium, represents a determinant of pathologic hypertrophy that leads to ventricular dysfunction. Therefore, to evaluate the potential benefit of the angiotensin converting enzyme (ACE) inhibitor quinapril in reversing the interstitial remodeling in spontaneously hypertensive rats (SHR) with established left ventricular hypertrophy (LVH), we treated 16-week-old male SHR with oral quinapril (average dose, 10 mg/kg body weight/day) for 20 weeks. Interstitial fibrosis was determined morphometrically using an automatic image analyzer. The amount of collagen was evaluated by measuring myocardial hydroxyproline concentration. Myocardial deposition of collagen molecules (types I, III, and IV) and other ECM components (fibronectin, laminin) was analyzed by immunohistochemical techniques using specific monoclonal antibodies. The activity of ACE was measured in left ventricular tissue by a fluorometric assay. In quinapril-treated SHR compared with 36-week-old untreated SHR and age- and sex-matched Wistar-Kyoto (WKY) controls, we found 1) a lesser degree of LVH and a lesser level of blood pressure, 2) a lesser degree of interstitial fibrosis, represented by less interstitial collagen volume fraction (5.73 +/- 0.45% v 3.42 +/- 0.28%, P < .05; WKY, 3.44 +/- 0.66%), 3) a lower hydroxyproline concentration (1.09 +/- 0.05 mumol/L/g dry weight/100 g body weight to 0.81 +/- 0.05 mumol/L/g dry weight/100 g body weight, P < .05; WKY, 0.96 +/- 0.06 mumol/L/g dry weight/100 g body weight), 4) a lesser presence of collagen fibers, and 5) a lesser presence of collagen IV, fibronectin, and laminin.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemistry in the differential diagnosis of serous effusions

The distinction between pleural mesothelioma (MS), reactive mesothelium (RM), and adenocarcinoma (AC) in serous effusions continues as a diagnostic problem in pathology. Immunohistochemistry can help, especially in surgical samples, but the optimum panel of antibodies has yet to be reported. The application of these antibodies to serous effusions has displayed variable results. The aim of this study was to evaluate the usefulness of eight monoclonal antibodies in the differential diagnosis of MS, RM, and AC in serous effusions.

Mast cells in chronic rejection of human renal allografts.

Abstract An increased number of mast cells (MCs) is found in renal specimens of patients with diseases associated with persistent chronic inflammation. MCs proliferation is partly dependent on the presence of T lymphocytes. Both chronic inflammation and T-lymphocytes are essential in the development of chronic rejection (CR), and probably for the infiltration of MCs. MC-derived products such as heparin, histamine, and serine proteases may be responsible for endothelial proliferation and excess collagen production by fibroblasts. In this study, a quantitative evaluation of the MCs infiltration in kidney allografts with CR is performed. The extent of renal fibrosis was analysed in samples stained with Masson’s trichrome. To evaluate the potential relationship between MCs and fibrosis in CR we analysed 30 kidneys with CR (25 from nephrectomies and 5 from autopsies). Ten transplanted kidneys obtained from patients died by causes not related with rejection were used as controls. CR was graded according to the Banff schema, which assesses the degree of vasculopathy, tubular atrophy, interstitial fibrosis and transplantation glomerulopathy. Giemsa-stained sections and immunohistochemistry using anti-MC tryptase and c-kit monoclonal antibodies were used to detect MCs. The mean number of MCs per 20 high-power fields (HPF) in the transplanted kidney with CR was 101.8±15.3 in the renal cortex and 46.60±6.52 in the medulla. MCs were significantly more numerous in CR with respect to normal kidneys, both in the cortex (P<0.01; Mann-Whitney U test) and in the medulla (P<0.01; Mann-Whitney U test). There was a positive correlation between the number of MCs and extent of fibrosis (P<0.01; Kruskal-Wallis one-way anova test) and tubular atrophy (P<0.01). These results suggest that MCs may play a role in the process of development of interstitial fibrosis in CR.

Susceptibility to apoptosis measured by MYC, BCL-2, and BAX expression in arterioles and capillaries of adult spontaneously hypertensive rats

Hypertension results in microvascular rarefaction or disappearance of microvessels. In the present study, we investigated the pathogenic role of apoptosis in hypertension-induced rarefaction of heart arterioles and capillaries of spontaneously hypertensive rats (SHR). Experiments were performed on hearts from 6-week-old, 16-week-old, and 30-week-old SHR (n = 30 rats) (SHR6, SHR16, SHR30). We used as controls 6-week-old, 16-week-old, and 30-week-old normotensive rats (WKY) (n = 30 rats) (WKY6, WKY16, WKY30). We analyzed the expression of c-myc, bcl-2, and bax and in situ end-labeling DNA fragmentation in vascular smooth muscle cells of arterioles and endothelial cells of arterioles and capillaries. Endothelial cells of capillaries and endothelial and smooth muscle cells of arterioles of hypertensive animals (SHR) express more Bax protein and Myc protein than their respective normotensive controls by margins that were statistically significant. The SHR30 group expressed the lowest levels of Bcl-2 protein by a margin that was statistically significantly different from WKY30. We did not find evidence of apoptosis in arterioles or capillaries on the basis of in situ end-labeling. However, our results indicated that alterations in the expression of members of the Bcl-2 family of proteins and Myc protein occurred in smooth muscle cells and endothelial cells of arterioles and capillaries of SHR. In conclusion, although evidence of apoptosis in arterioles and capillaries was not found by in situ end-labeling, our findings suggest that in hypertension they may have a higher susceptibility to apoptosis, and therefore rarefaction may be a consequence of apoptosis.