Alaa Afify

Adipose Tissue Dysregulation in Patients with Metabolic Syndrome

CONTEXT The metabolic syndrome (MetS) is associated with increased risk of diabetes and cardiovascular disease (CVD). Numerous groups have shown increased circulating biomarkers of inflammation in MetS. However, there are scanty data on the cellular sources contributing to this low-grade inflammation. OBJECTIVE The aim of this study was to determine the role of sc adipose tissue (SAT) biology in nascent MetS without concomitant diabetes or CVD. PATIENTS AND METHODS Subjects with MetS and controls were recruited after informed consent. Fasting blood was collected, and SAT was obtained by biopsy. RESULTS Circulating biomarkers of inflammation and insulin resistance, high-sensitivity C-reactive protein (hsCRP), IL-6, IL-1β, leptin, serum amyloid A, and retinol-binding protein-4 (RBP-4) concentrations were significantly higher in the MetS subjects than controls, whereas adiponectin concentrations were lower. In SAT, leptin, RBP-4, CRP, serum amyloid A, plasminogen activator inhibitor-1, IL-1, IL-6, IL-8, and monocyte chemotactic protein-1 (MCP-1) levels were significantly higher in MetS than controls. These differences except for RBP-4 persisted after adjusting for waist circumference. In addition, there were significantly increased numbers of macrophages infiltrating the SAT of MetS and increased numbers of crown-like structures compared with controls. hsCRP correlated positively with homeostasis model assessment and SAT MCP-1 and negatively with adiponectin. Homeostasis model assessment correlated positively with plasminogen activator inhibitor-1, RBP-4, and SAT MCP-1. CONCLUSIONS We make the novel observation that SAT of MetS has increased macrophage recruitment with cardinal crown-like structure features and contributes to the increased cellular inflammation that produces increased levels of biomarkers that are correlated with both insulin resistance and low-grade inflammation. These aberrations could contribute to the progression of MetS and the increased risk for diabetes and CVD.

Human C-reactive protein promotes oxidized low density lipoprotein uptake and matrix metalloproteinase-9 release in Wistar rats

C-reactive protein (CRP) is present in the atherosclerotic plaques and appears to promote atherogenesis. Intraplaque CRP colocalizes with oxidized low density lipoprotein (OxLDL) and macrophages in human atherosclerotic lesions. Matrix metalloproteinase-9 (MMP-9) has been implicated in plaque rupture. CRP promotes OxLDL uptake and MMP induction in vitro; however, these have not been investigated in vivo. We examined the effect of CRP on OxLDL uptake and MMP-9 production in vivo in Wistar rats. CRP significantly increased OxLDL uptake in the peritoneal and sterile pouch macrophages compared with human serum albumin (huSA). CRP also significantly increased intracellular cholesteryl ester accumulation compared with huSA. The increased uptake of OxLDL by CRP was inhibited by pretreatment with antibodies to CD32, CD64, CD36, and fucoidin, suggesting uptake by both scavenger receptors and Fc-γ receptors. Furthermore, CRP treatment increased MMP-9 activity in macrophages compared with huSA, which was abrogated by inhibitors to p38 mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK), and nuclear factor (NF)-κB but not Jun N-terminal kinase (JNK) before human CRP treatment. Because OxLDL uptake by macrophages contributes to foam cell formation and MMP release contributes to plaque instability, this study provides novel in vivo evidence for the role of CRP in atherosclerosis.

Knockout of Toll-Like Receptor-2 Attenuates Both the Proinflammatory State of Diabetes and Incipient Diabetic Nephropathy

Objective—Type 1 diabetes (T1DM) is a proinflammatory state and confers an increased risk for vascular complications. Toll-like receptors (TLR) could participate in diabetic vasculopathies. Whether TLR activation contributes to the proinflammatory state of T1DM and the pathogenesis of diabetic nephropathy remains unknown. Methods and Results—We induced T1DM in TLR2 knockout mice (TLR2−/−) and wild-type littermates (C57BL/6J-WT) using streptozotocin (STZ). Fasting blood, peritoneal macrophages, and kidneys were obtained for flow cytometry, Western blot, microscopy, and cytokine assays at 6 and 14 weeks after induction of diabetes. Macrophage TLR2 expression and MyD88-dependent signaling were increased in diabetic mice (WT+STZ) compared with nondiabetic WT mice. These biomarkers were attenuated in diabetic TLR2−/− macrophages. WT+STZ mice showed increased kidney:body weight ratio due to cell hypertrophy, increased albuminuria, decreased kidney nephrin, podocin, and podocyte number and increased transforming growth factor-&bgr; and laminin compared with WT mice. Nephrin, podocin, and podocyte number and effacement were restored, and transforming growth factor-&bgr; and laminin levels were decreased in TLR2−/−+ STZ mice kidneys versus WT+STZ. Peritoneal and kidney macrophages were predominantly M1 phenotype in WT+STZ mice; this was attenuated in TLR2−/−+STZ mice. Conclusion—These data support a role for TLR2 in promoting inflammation and early changes of incipient diabetic nephropathy, in addition to albuminuria and podocyte loss.

Role of CD44s and CD44v6 on human breast cancer cell adhesion, migration, and invasion

The interaction between the transmembrane receptor CD44 on epithelial tumor cells and its ligand hyaluronan in the surrounding extracellular matrix is important in tumor progression and metastasis. CD44 is encoded by a single 20-exon gene and expressed in standard form (CD44s), as well as a myriad of CD44 variants (CD44v) generated by alternative splicing of the CD44 mRNA. Previously, we demonstrated that hyaluronan (HA) production is increased at tumor-stroma interface in invasive and metastatic human breast cancers when compared with benign or premalignant lesions. We hypothesize that CD44 expression on breast cancer cells is a major contributing factor to cell adhesion, migration and invasion. To evaluate this hypothesis we examined the effects of 3 distinct anti-CD44s and 2 anti-CD44v6 monoclonal antibodies on breast cancer cell lines that expressed high and low CD44s and CD44v6. Using these antibodies we assessed the role of CD44 in cell adhesion, cell motility, and cell invasion using immobilized HA-coated wells, wound healing assays, and modified Boyden chamber respectively. Our results showed that anti-CD44s could inhibit breast cancer cell adhesion, motility and invasion, while anti-CD44v6 inhibits cell motility. In conclusion, our data suggests that CD44s is involved in breast cancer cell adhesion, motility and invasion through interaction with HA but CD44v6 is involved only in cell motility. Furthermore we concluded that antibodies against different epitopes on CD44 mediate distinct functional effects on breast cancer cells.

Temporal variation in the distribution of hyaluronic acid, CD44S, and CD44v6 in the human endometrium across the menstrual cycle

Tissues undergoing rapid growth and regeneration contain hyaluronic acid (HA) as a prominent component of the extracellular matrix. The physiologic role of HA is partly mediated by its relationship with CD44, its major cell surface receptor. Given the extensive remodeling of the endometrium during the menstrual cycle, the authors sought to determine whether these changes are related to the levels of HA, CD44s, and CD44v6 in the endometrium. Archival paraffinembedded cell blocks from 10 cases of proliferative endometrium and 20 cases of secretory endometrium were retrieved from the surgical pathology files. Specimens from the secretory phase were subdivided into three categories: early secretory (day 15–18), mid-secretory (day 19–23), and late secretory (day 24–28). All cases were stained for hyaluronic acid, CD44s, and CD44v6. Sections from umbilical cord, tonsil, and squamous cell carcinoma served as positive controls for HA, CD44s, and CD44v6, respectively. Positive staining was defined as droplet to diffuse intracytoplasmic or extracellular staining for HA and uniform membranous staining for CD44. During the proliferative phase, the endometrial glands and the stroma were both negative for CD44s and CD44v6 in all cases. In the secretory phase, the endometrial glands were negative for CD44s in all cases, but CD44v6 was expressed in 12 (60%) of cases. In contrast, the stromal cells expressed CD44s in 18 (90%) cases and were negative for CD44v6 in all cases. HA staining was present in the endometrial stroma throughout the menstrual cycle but was most intense (3+) and diffuse during the midsecretory phase. There was perivascular staining for HA throughout the cycle; it was most intense adjacent to the spiral arterioles in the secretory phase. These data indicate temporal and geographic differences in HA and CD44 staining in the endometrium in concert with the menstrual cycle. The timing of peak staining of HA and CD44s in the stroma and the upregulation of CD44v6 in secretory glands are coincident with the period in which the endometrium is most receptive to embryo implantation. Whether these changes are mere hormonal consequences or actually help modulate the cyclical changes in the endometrium warrants further study.

Expression of CD44s, CD44v6, and hyaluronan across the spectrum of normal-hyperplasia-carcinoma in breast.

BackgroundThe interaction between transmembrane receptors on epithelial tumor cells and the surrounding extracellular matrix molecules is important in tumor progression and metastasis. This interaction is best exemplified by the relationship of the receptor CD44 and the extracellular matrix component hyaluronan (HA). This study seeks to evaluate the expression and the correlation of CD44s, CD44v6, and HA in normal, hyperplastic, and malignant breast epithelium and stroma. Materials and MethodsArchival paraffin-embedded tissue from cases of normal breast tissue (n=10), intraductal hyperplasia without atypia (n=13), ductal carcinoma in situ (DCIS) (n=24), stage I infiltrating ductal carcinoma (n=28), stage II infiltrating ductal carcinoma (n=31), and their corresponding positive lymph nodes were retrieved from the surgical pathology files. Tissue sections were evaluated for the expression of CD44s, CD44v6, and HA in the epithelial and stromal cells by immunohistochemistry. ResultsDuctal epithelial cells and myoepithelial cells expressed CD44s in all cases of normal and benign breast tissue. The expression of CD44s in breast epithelium progressively decreased with increasing deviation from normal histology: 83% in DCIS, 46% in stage I ductal carcinoma and 26% in stage II ductal carcinoma. The reverse trend was observed for CD44v6 in ductal epithelium: 0% in normal breast, 15% in intraductal hyperplasia, 100% in DCIS, 82% in stage I infiltrating ductal carcinoma, 94% in stage II carcinoma, and 100% of metastatic carcinoma in the lymph nodes. HA was noted exclusively in the stroma but not in the epithelial cells. HA was faintly expressed in the intralobular stroma of normal breast tissue, confined to a narrow faint band adjacent to intraductal hyperplasia and localized to a broad well-defined band around DCIS. Stromal HA staining was more diffuse and intense in infiltrating carcinomas and was particularly pronounced surrounding the metastatic deposits in lymph nodes. ConclusionsThis study demonstrates decreased expression of CD44s accompanied by increased expression of CD44v6 and increased stromal HA in breast cancer. These findings suggest that CD44s, CD44v6, and HA play complementary roles in the development and progression of breast cancer.

Expression of CD44s and CD44v6 in lung cancer and their correlation with prognostic factors

Background CD44, a transmembrane glycoprotein receptor, plays a major role in tumor progression and metastasis. Objective: To evaluate the expression of CD44 standard (CD44s) and its variant 6 (CD44v6) in normal and neoplastic lung tissue and correlate it with prognostic factors in lung cancer. Methods The study included 52 non-small cell lung carcinomas (NSCLC) (21 squamous cell carcinomas and 31 adenocarcinomas), 15 small cell lung carcinomas (SCLC) and 8 carcinoid tumors. Expression of CD44s and CD44v6 was evaluated by immunohistochemistry and correlated with lung cancer prognostic factors. Results All squamous cell carcinomas expressed both CD44s and CD44v6. Adenocarcinomas expressed CD44s in 39% of cases and CD44v6 in 45%. Carcinoid tumors expressed only CD44s in 88% of cases. All SCLCS were negative for both CD44s and CD44v6. A restricted panel consisting of CD44s and CD44v6 will discriminate NSCLC from SCLC with a sensitivity of 67% and a specificity of 100%. In adenocarcinoma CD44s expression was significantly correlated with lymph node metastases (p=0.007) while CD44v6 expression was more significantly associated with tumor size (p=0.0032). Conclusions CD44s and CD44v6 are expressed in certain types of lung cancer. In adenocarcinoma CD44s and CD44v6 expression is significantly correlated with lymph node metastases and tumor size.

Diagnostic Utility of GLUT-1 Expression in the Cytologic Evaluation of Serous Fluids

OBJECTIVE To evaluate the extent to which adenocarcinomas in body cavity fluids express GLUT-1 in comparison to currently available markers for adenocarcinomas. STUDY DESIGN Archival paraffin-embedded cell blocks of serous fluids from 25 cases of benign effusions containing reactive mesothelial cells and 39 cases of malignant effusions with metastatic adenocarcinoma (11 ovarian, 11 pulmonary, 9 gastrointestinal and 8 breast) were retrieved from the surgical pathology files. All cases were stained with antibodies for GLUT-1, Ber-Ep4, B72.3 and CEA. Positive staining was defined as distinct linear membrane staining for GLUT-1 and Ber-EP4, cytoplasmic staining for CEA, and cytoplasmic or membrane staining for B72.3. Strong staining in at least 10% of the tumor cells was required in order to consider the case positive for the particular marker. RESULTS GLUT-1 was expressed in 72% (28 of 39) of cases of malignant effusions: 100% (11 of 11) from the ovary, 91% (10 of 11) from the lung, 67% (6 of 9) from the gastrointestinal tract and 12% (1 of 8) from the breast. None (0 of 25) of the benign effusions expressed GLUT-1. Malignant effusions expressed CEA in 74% (29 of 39), Ber-Ep4 in 85% (33 of 39), and B72.3 in 62% (24 of 39). Benign effusions expressed CEA in 3 cases and B72.3 in 2 cases. CONCLUSION GLUT-1 is a useful marker that can be applied to cytologic specimens. It can be used as a reliable component of an antibody panel to distinguish reactive mesothelial cells from metastatic adenocarcinoma in particular adenocarcinomas of body cavity effusions, in particular adenocarcinomas of ovarian and pulmonary origin.

Impact of Pulmonary Vein Cryoballoon Ablation on Bronchial Injury

There is a paucity of data on the mechanisms of cough and hemoptysis that sometimes ensue from cryoballoon ablation of pulmonary veins (Cryo‐PV). This study specifically examined the impact of ultra‐cold (≤−60 °C, 3 minutes), prolonged (>−55 °C, 6 minutes), and conventional (>−55 °C, 3 minutes) Cryo‐PV on lung/bronchial injury.

GLUT-1 is preferentially expressed in atypical endometrial hyperplasia and endometrial adenocarcinoma.

The facilitative transport of monosaccharides in human cells is accomplished by a family of transmembrane proteins, GLUT-1 to GLUT-7, that differ in their tissue distribution, affinities for specific monosaccharides, and physiologic regulation. GLUT-1, a high-affinity glucose transporter, is normally expressed in erythrocytes, the perineurium of peripheral nerves, and capillary endothelial cells of the blood–brain barrier. Although the aberrant expression of GLUT-1 has been reported in a wide spectrum of epithelial malignancies, its possible correlation with the malignant transformation of endometrial epithelium has not been clearly established. The purpose of this study was to evaluate the extent to which benign, hyperplastic, atypical, and malignant endometrial epithelia express GLUT-1. The authors examined the IHC expression of GLUT-1 in cases of proliferative endometrium (n=12), secretory endometrium (n=10), endometrial polyps (n=10), adenomyosis (n=18), simple hyperplasia (n=14), complex hyperplasia without atypia (n=17), complex hyperplasia with atypia (n=17), and adenocarcinoma (n=31). Positive staining was defined as distinct, linear membrane staining, particularly at cell–cell borders. Cells that showed only cytoplasmic staining were considered negative. The percentages of positive cells and staining intensity were assessed in a semiquantitative fashion and scored (1+ to 3+). All cases from proliferative endometrium, secretory endometrium, adenomyosis, and simple hyperplasia and 90% (9/10 cases) of the endometrial polyps were negative for GLUT-1. GLUT-1 was expressed in 24% (4/17 cases) of complex hyperplasia without atypia, 71% (12/17 cases) of complex hyperplasia with atypia, and 90% (28/31 cases) of adenocarcinomas. The extent of staining ranged from occasional positive foci to extensive multifocal staining. GLUT-1 positivity increased in intensity as the distance of tumor cells to stroma increased. The authors conclude that GLUT-1 is preferentially expressed in complex hyperplasia with atypia and in adenocarcinoma and that GLUT-1 immunostaining is useful in distinguishing benign hyperplasia from hyperplasia strongly associated with malignancy. GLUT-1-mediated glucose transport may allow hypoxic tumor cells distant from stromal blood vessels to survive through glycolysis. These data suggest that the expression of GLUT-1 transporter may be closely related to the malignant transformation of epithelial endometrial tumors by supporting their increased need for glucose metabolism.