Hearts lacking plasma membrane KATP channels display changes in basal aerobic metabolic substrate preference and AMPK activity.

Cardiac ATP-sensitive K+ (KATP) channels couple changes in cellular metabolism to membrane excitability and are activated during metabolic stress, although under basal aerobic conditions, KATP channels are thought to be predominately closed. Despite intense research into the roles of KATP channels during metabolic stress, their contribution to aerobic basal cardiac metabolism has not been previously investigated. Hearts from Kir6.2+/+ and Kir6.2-/- mice were perfused in working mode, and rates of glycolysis, fatty acid oxidation, and glucose oxidation were measured. Changes in activation/expression of proteins regulating metabolism were probed by Western blot analysis. Despite cardiac mechanical function and metabolic efficiency being similar in both groups, hearts from Kir6.2-/- mice displayed an approximately twofold increase in fatty acid oxidation and a 0.45-fold reduction in glycolytic rates but similar glucose oxidation rates compared with hearts from Kir6.2+/+ mice. Kir6.2-/- hearts also possessed elevated levels of activated AMP-activated protein kinase (AMPK), higher glycogen content, and reduced mitochondrial density. Moreover, activation of AMPK by isoproterenol or diazoxide was significantly blunted in Kir6.2-/- hearts. These data indicate that KATP channel ablation alters aerobic basal cardiac metabolism. The observed increase in fatty acid oxidation and decreased glycolysis before any metabolic insult may contribute to the poor recovery observed in Kir6.2-/- hearts in response to exercise or ischemia-reperfusion injury. Therefore, KATP channels may play an important role in the regulation of cardiac metabolism through AMPK signaling.NEW & NOTEWORTHY In this study, we show that genetic ablation of plasma membrane ATP-sensitive K+ channels results in pronounced changes in cardiac metabolic substrate preference and AMP-activated protein kinase activity. These results suggest that ATP-sensitive K+ channels may play a novel role in regulating metabolism in addition to their well-documented effects on ionic homeostasis during periods of stress.

The mechano-sensitivity of cardiac ATP-sensitive potassium channels is mediated by intrinsic MgATPase activity.

Cardiac ATP-sensitive K+ (KATP) channel activity plays an important cardio-protective role in regulating excitability in response to metabolic stress. Evidence suggests that these channels are also mechano-sensitive and therefore may couple KATP channel activity to increased cardiac workloads. However, the molecular mechanism that couples membrane stretch to channel activity is not currently known. We hypothesized that membrane stretch may alter the intrinsic MgATPase activity of the cardiac KATP channel resulting in increased channel activation. The inside-out patch-clamp technique was used to record single-channel and macroscopic recombinant KATP channel activity in response to membrane stretch elicited by negative pipette pressure. We found that stretch activation requires the presence of the SUR subunit and that inhibition of MgATPase activity with either the non-hydrolysable ATP analog AMP-PNP or the ATPase inhibitor BeFx significantly reduced the stimulatory effect of stretch. We employed a point mutagenic approach to determine that a single residue (K1337) in the hairpin loop proximal to the major MgATPase catalytic site in the SUR2A subunit is responsible for the difference in mechano-sensitivity between SUR2A and SUR1 containing KATP channels. Moreover, using a double cysteine mutant substitution in the hairpin loop region revealed the importance of a key residue-residue interaction in this region that transduces membrane mechanical forces into KATP channel stimulation via increases in channel MgATPase activity. With respect to KATP channel pharmacology, glibenclamide, but not glicalizide or repaglinide, was able to completely inhibit KATP channel mechano-sensitivity. In summary, our results provide a highly plausible molecular mechanism by which mechanical membrane forces are rapidly converted in changes in KATP channel activity that have implications for our understanding of cardiac KATP channels in physiological or pathophysiological settings that involve increased workload.

Diagnostic Utility of Thyroid Transcription Factor-1 in Ovarian Carcinoma and Its Relationship With Clinicopathologic Prognostic Parameters.

Although thyroid transcription factor-1 (TTF-1) is considered a relatively sensitive and specific marker for lung and thyroid neoplasms, it can occasionally be expressed in other tumors. Few immunohistochemical studies have been conducted on TTF-1 expression in ovarian carcinomas with discrepant results. To date, only 1 study compared different TTF-1 clones in ovarian carcinoma. This study is designed to evaluate the expression of TTF-1 clones in ovarian carcinomas and investigate TTF-1 association with clinicopathologic prognostic parameters. A retrospective immunohistochemical study was conducted on 62 primary ovarian carcinomas and 15 normal ovarian tissues using 2 clones of TTF-1 antibody (SPT24 and 8G7G3/1). Nuclear expression of SPT24 and 8G7G3/1 clones of TTF-1 was detected in 17.7% and 3.2% of ovarian carcinomas, respectively. Positive cytoplasmic immunostaining of clone SPT24 was detected in 1.6% of cases. In contrast, normal ovarian tissue showed negative expression of both clones. A highly significant difference was observed between both clones regarding their sensitivity in ovarian carcinomas (P=0.004). A significant inverse relationship was observed between TTF-1 (SPT24 clone) expression and tumor stage (P=0.022). TTF-1 expression is not exclusive to lung and thyroid tissue. It is expressed in ovarian carcinomas where clone SPT24 is more sensitive than clone 8G7G3/1. TTF-1 might be of diagnostic utility in evaluating neoplasms of unknown primary origin as well as adenocarcinomas involving the lung in patients with a history of a gynecologic malignancy. Moreover, TTF-1 expression might be a good prognostic factor in ovarian carcinoma.

Combined use of COX-1 and VEGF immunohistochemistry refines the histopathologic prognosis of renal cell carcinoma.

The course of RCC is asymptomatic, resulting in 25-30% of patients presenting with metastatic disease at time of diagnosis. The development of novel agents targeting angiogenesis and signal transduction pathways has improved patient outcomes. Role of cyclooxygenase in cancer development has been the subject of close scrutiny. COX-1 has been recognized to be involved in regulation of angiogenesis. To date, no immunohistochemical studies have been performed to assess the possible association between COX-1 and VEGF in RCC. This study is designed to evaluate the relationship between these two proteins in RCC. Also, the relationship between their combined immunohistochemical expression and different clinicopathological prognostic parameters in RCC is investigated. Immunohistochemical expression of COX-1 and VEGF was evaluated retrospectively on 64 cases of primary RCC including: 45 clear cell carcinoma, 12 papillary carcinoma and 7 of chromophope carcinoma. High COX-1 expression was detected in 62.5% of RCCs with a significant association with tumor grade (P=0.028), and highly significant relationship with tumor size and stage (P=0.001). There was a highly significant relationship between the VEGF score and tumor size (P=0.001), and stage (P=0.006). There was a positive correlation between COX-1 and VEGF expression score (P=0.001). Combined expression of both markers predicts high stage tumors (stage III/IV). Immunohistochemical expression of COX-1 and VEGF is associated with poor prognostic parameters in RCC. Their combined expression has a beneficial role in prediction of high stage tumors (III/IV).

Molecular determinants of ATP-sensitive potassium channel MgATPase activity: diabetes risk variants and diazoxide sensitivity.

ATP-sensitive K(+) (KATP) channels play an important role in insulin secretion. KATP channels possess intrinsic MgATPase activity that is important in regulating channel activity in response to metabolic changes, although the precise structural determinants are not clearly understood. Furthermore, the sulfonylurea receptor 1 (SUR1) S1369A diabetes risk variant increases MgATPase activity, but the molecular mechanisms remain to be determined. Therefore, we hypothesized that residue-residue interactions between 1369 and 1372, predicted from in silico modelling, influence MgATPase activity, as well as sensitivity to the clinically used drug diazoxide that is known to increase MgATPase activity. We employed a point mutagenic approach with patch-clamp and direct biochemical assays to determine interaction between residues 1369 and 1372. Mutations in residues 1369 and 1372 predicted to decrease the residue interaction elicited a significant increase in MgATPase activity, whereas mutations predicted to possess similar residue interactions to wild-type (WT) channels elicited no alterations in MgATPase activity. In contrast, mutations that were predicted to increase residue interactions resulted in significant decreases in MgATPase activity. We also determined that a single S1369K substitution in SUR1 caused MgATPase activity and diazoxide pharmacological profiles to resemble those of channels containing the SUR2A subunit isoform. Our results provide evidence, at the single residue level, for a molecular mechanism that may underlie the association of the S1369A variant with type 2 diabetes. We also show a single amino acid difference can account for the markedly different diazoxide sensitivities between channels containing either the SUR1 or SUR2A subunit isoforms.

Relationship between osteopontin and ß-catenin immunohistochemical expression and prognostic parameters of colorectal carcinoma.

Previous studies on the prognostic value of osteopontin (OPN) and ß-catenin in colorectal carcinoma (CRC) revealed conflicting results. To date, only two immunohistochemical studies investigated their association in CRC with discrepant results. Moreover, the relevance of their co-expression to clinicopathological parameters was not previously reported. This study was designed to investigate the relationship between these markers and prognostic parameters in CRC and study further the relationship between them. Immunohistochemical expression of OPN and ß-catenin was evaluated in 72 CRCs. Cytoplasmic OPN was detected in 45.83% of CRCs while normal mucosa was immunonegative. Strong continuous membranous ß-catenin was present in normal mucosa. However, abnormal membranous, nuclear and cytoplasmic expressions were observed in 36.11%, 31.94% and 52.78% of CRCs, respectively. A highly significant relationship was detected between each of OPN and nuclear ß-catenin expression and lymph node metastasis (P = 0.0001 and 0.004 respectively), depth of invasion (P = 0.001 and 0.004 respectively), TNM stages (P = 0.0001 and 0.001 respectively) and Dukes' stages (P = 0.0001 and 0.004 respectively). A significant association was found between OPN and distant metastases. A strong agreement was observed between OPN and nuclear ß-catenin (kappa = 0.656). A highly significant relationship was found between their co-expression and poor prognostic parameters. OPN overexpression and nuclear ß-catenin expression appeared to be associated with unfavorable prognostic factors in CRC. A direct relationship was observed between them. Further understanding their role in colorectal carcinogenesis as well as targeting the interaction between them might be effective in the future development of therapeutic agents for CRC patients.

Immunohistochemical expression of CD117 and vascular endothelial growth factor in retinoblastoma: possible targets of new therapies.

CD117 (C-kit) is thought to play an important role in tumourigenesis. There are limited data in the literature concerning C-kit expression in retinoblastoma. To date, no immunohistochemical studies have been performed to assess the possible association of C-kit with vascular endothelial growth factor (VEGF) in retinoblastoma. This study was designed to investigate C-kit and VEGF immunoexpression in retinoblastoma, their relationship with prognostic parameters as well as the correlation between them. A prospective immunohistochemical study was conducted on 56 retinoblastoma cases. Patients who had received preoperative chemotherapy were excluded. Positive C-kit and VEGF immunoreactivity was observed in 48.2% and 76.8% of retinoblastoma cases respectively. No C-kit immunostaining was seen in the adjacent uninvolved retina. However, VEGF expression was detected within its vasculature. Retinoblastomas with combined pattern of tumour growth revealed a highly significant positive C-kit expression (P = 0.002) compared to cases with endophytic or exophytic growths. Also, positive C-kit expression was statistically higher in cases with optic nerve invasion (P = 0.001) and choroidal invasion (P ≤ 0.01) compared to negative cases. A highly significant positive VEGF expression was detected in cases with optic nerve invasion (P = 0.013) compared to negative cases. Moreover, a highly significant positive correlation was detected between C-kit and VEGF expression (P = 0.006). C-kit is a feature of more aggressive retinoblastomas, with increased expression in tumours spreading beyond the retina. Moreover, VEGF is vastly expressed in retinoblastoma and is associated with optic nerve invasion. Both C-kit and VEGF may represent potential therapeutic targets for retinoblastomas.

Association of Fascin and matrix metalloproteinase-9 expression with poor prognostic parameters in breast carcinoma of Egyptian women.

BACKGROUND: The effects of fascin on cell invasiveness involve changes in cell motility and matrix metalloproteinase-9 (MMP-9) activity. Previous studies on the prognostic value of fascin and MMP-9 in breast carcinoma revealed conflicting results. To date, no immunohistochemical studies have been performed to assess the possible association between them in breast carcinoma. This study is designed to correlate their expression with prognostic parameters in breast carcinoma and assess the relationship between them. METHODS: Immunohistochemical expression of fascin and MMP-9 was evaluated semi quantitatively in 67 cases of breast carcinoma regarding the percentage of positive cells. Chi square test and Fisher's exact test were used to examine the relationship between categorical variables. Kappa statistics was used to compute the measure of agreement between two investigational methods. RESULTS: Fascin and MMP-9 expressions were detected in 43.28% and 50.75% of breast carcinomas (respectively). Regarding the normal breast tissue, fascin expression was observed in myoepithelial cells and luminal cells of few ducts and acini. However, normal tissue showed negative MMP-9 expression. A significant relationship was observed between fascin and MMP-9 expression and lymph node metastases (p = 0.001 and 0.002 respectively), advanced tumor stage (p = 0.004 and 0.005 respectively), estrogen receptor negative (p = 0.002 and 0.005 respectively), progesterone receptor negative (p = 0.001 and 0.003 respectively) hormonal status and molecular subtypes (p = 0.0007 and 0.014 respectively). A significant strong agreement was detected between fascin and MMP-9 expression (p = 0.0001). More intense immunostaining of fascin and MMP-9 was observed at the invasive fronts compared with other areas of the tumor. Moreover, a significant moderate agreement between fascin and MMP-9 was found regarding the site of predominant intensity. CONCLUSION: Fascin and MMP-9 proteins are associated with parameters of poor prognosis in breast cancer. The significant strong agreement between the two markers supports the role of fascin in cell invasiveness by activating matrix proteases besides increasing cell motility. Both proteins may represent potential therapeutic targets for patients with breast cancer especially those with hormone receptor-negative status. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1421167695121127.

Intracellular long-chain acyl CoAs activate TRPV1 channels.

TRPV1 channels are an important class of membrane proteins that play an integral role in the regulation of intracellular cations such as calcium in many different tissue types. The anionic phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) is a known positive modulator of TRPV1 channels and the negatively charged phosphate groups interact with several basic amino acid residues in the proximal C-terminal TRP domain of the TRPV1 channel. We and other groups have shown that physiological sub-micromolar levels of long-chain acyl CoAs (LC-CoAs), another ubiquitous anionic lipid, can also act as positive modulators of ion channels and exchangers. Therefore, we investigated whether TRPV1 channel activity is similarly regulated by LC-CoAs. Our results show that LC-CoAs are potent activators of the TRPV1 channel and interact with the same PIP2-binding residues in TRPV1. In contrast to PIP2, LC-CoA modulation of TRPV1 is independent of Ca2+i, acting in an acyl side-chain saturation and chain-length dependent manner. Elevation of LC-CoAs in intact Jurkat T-cells leads to significant increases in agonist-induced Ca2+i levels. Our novel findings indicate that LC-CoAs represent a new fundamental mechanism for regulation of TRPV1 channel activity that may play a role in diverse cell types under physiological and pathophysiological conditions that alter fatty acid transport and metabolism such as obesity and diabetes.

Endothelin-1 and its A and B receptors: are they possibly involved in vitiligo?

Endothelin-1 (ET-1), expressed by keratinocytes, has paracrine effects on melanocytes. The endothelin 1-axis [ET-1, endothelin A receptor (ETAR) and endothelin B receptor (ETBR)] is thought to play a role in the depigmentation process occurring in vitiligo, with no studies on the cutaneous protein expression of this axis in the disease. The aim of the present study was to compare the expression of ET-1 axis in lesional and perilesional normal epidermis of vitiligo patients with healthy controls. Ten patients with non-segmental stable vitiligo and ten healthy controls were included. Skin biopsies from all subjects were studied immunohistochemically for ET-1, ETAR and ETBR expression. No significant difference was detected in the rate of expression and the degree of staining of ET-1 axis in controls compared with each of lesional vitiligo and perilesional normal epidermis (P>0.05). There was no statistically significant difference between lesional vitiligo and perilesional normal epidermis regarding to the rates of ET-1, ETAR and ETBR expression (P=0.82, P=0.5 and P=0.99, respectively). Semi-quantitative analysis of ETAR revealed higher staining grades in lesional compared with perilesional normal epidermis, with a statistically significant difference (P=0.04). There was no statistically significant difference between the two groups regarding the staining grades of ET-1 and ETBR (P>0.05 for both markers). A highly significant positive correlation was found between ET-1 and ETAR (r =0.99, P<0.05) and between ET-1 and ETBR (r=0.87, P<0.05). The study demonstrated unaltered expression of ET-1 axis in keratinocytes in lesional vitiligo and perilesional normal epidermis. Additional studies on the differential expression of this axis in keratinocytes and melanocytes are therefore required.

Phosphorylation status of matrix metalloproteinase 2 in myocardial ischaemia-reperfusion injury.

OBJECTIVE: To investigate whether alterations in the phosphorylation status of matrix metalloproteinase 2 (MMP-2) in the heart may be protective in the setting of ischaemia-reperfusion (IR) injury. DESIGN: In-vitro heart function and biochemical research study. SETTING: University basic science laboratory. INTERVENTIONS: Male Sprague-Dawley rats, weighing 250-350 g. Isolated rat hearts were perfused at constant pressure either aerobically for 75 min or subjected to 20 min of global, no-flow ischaemia followed by 30 min of reperfusion. MAIN OUTCOME MEASURES: Heart mechanical function, MMP-2 activity and troponin I levels. RESULTS: The serine/threonine phosphatase inhibitor okadaic acid (OA) improved the recovery of mechanical function compared with control IR hearts and prevented the loss of troponin I. OA significantly reduced protein phosphatase 2A, but not protein phosphatase 1, activity in perfused hearts. IR stimulated the activation and release of MMP-2 into the coronary effluent in the first 2 min of reperfusion. This was accompanied by a decrease in the remaining activity and protein level of MMP-2 in heart tissue determined at the end of the reperfusion. OA did not alter the IR-stimulated release of MMP-2 into the coronary effluent, but reduced the decrease in MMP-2 in reperfused hearts. The immunoprecipitation of heart homogenates using anti-phosphoserine antibody showed that MMP-2 is phosphorylated. The dephosphorylation of MMP-2 by alkaline phosphatase treatment of homogenates prepared from IR hearts treated with OA significantly increased MMP-2 activity. CONCLUSIONS: These results suggest that the phosphorylation status of MMP-2 is important in its contribution to myocardial IR injury.

The molecular genetics of sulfonylurea receptors in the pathogenesis and treatment of insulin secretory disorders and type 2 diabetes.

Sulfonylurea receptors (SURs) form an integral part of the ATP-sensitive potassium (K(ATP)) channel complex that is present in most excitable cell types. K(ATP) channels couple cellular metabolism to electrical activity and provide a wide range of cellular functions including stimulus secretion coupling in pancreatic ß cells. K(ATP) channels are composed of SURs and inward rectifier potassium channel (Kir6.x) subunits encoded by the ABCC8/9 and KCNJ8/11 genes, respectively. Recent advances in the genetics, molecular biology, and pharmacology of SURs have led to an increased understanding of these channels in the etiology and treatment of rare genetic insulin secretory disorders. Furthermore, common genetic variants in these genes are associated with an increased risk for type 2 diabetes. In this review we summarize the molecular biology, pharmacology, and physiology of SURs and K(ATP) channels, highlighting recent advances in their genetics and understanding of rare insulin secretory disorders and susceptibility to type 2 diabetes.

Matrix metalloproteinase inhibitor properties of tetracyclines: therapeutic potential in cardiovascular diseases.

Matrix metalloproteinases (MMPs) are a family of proteases best known for their capacity to proteolyse several proteins of the extracellular matrix. Their increased activity contributes to the pathogenesis of several cardiovascular diseases. MMP-2 in particular is now considered to be also an important intracellular protease which has the ability to proteolyse specific intracellular proteins in cardiac muscle cells and thus reduce contractile function. Accordingly, inhibition of MMPs is a growing therapeutic aim in the treatment or prevention of various cardiovascular diseases. Tetracyclines, especially doxycycline, have been frequently used as important MMP inhibitors since they inhibit MMP activity independently of their antimicrobial properties. In this review we will focus on the intracellular actions of MMPs in some cardiovascular diseases including ischemia and reperfusion (I/R) injury, inflammatory heart diseases and septic shock; and explain how tetracyclines, as MMP inhibitors, have therapeutic actions to treat such diseases. We will also briefly discuss how MMPs can be intracellularly regulated and activated by oxidative stress, thus cleaving several important proteins inside cells. In addition to their potential therapeutic effects, MMP inhibitors may also be useful tools to understand the biological consequences of MMP activity and its respective extra- and intracellular effects.

Immunohistochemical assessment of angiogenesis and vascular endothelial growth factor expression in cutaneous lichen planus: relation to the degree of inflammation.

Neo-angiogenesis is reported to be essential in the pathogenesis of oral lichen planus (LP). We aimed to investigate angiogenesis through CD34 staining and vascular endothelial growth factor (VEGF) expression in cutaneous LP lesions and to evaluate the relation of these markers with the degree of inflammation. Thirty patients with cutaneous LP and 10 healthy controls were included. Skin biopsies from all subjects were studied immunohistochemically for microvessel density (MVD) by CD34 staining and for VEGF expression. Relation of these parameters with the grade of inflammation was evaluated. The mean MVD was significantly higher in patients than controls (32.60, 95% CI: 27.71-37.49 vs. 9.60, 95% CI: 6.86-12.34; t = 5.43; P < 0.001). Positive VEGF expression was detected at a significantly higher rate in LP patients compared with controls (76.7% vs. 30.0%, OR (95% CI) 7.67(1.56-37.80), P < 0.05). Patients with positive VEGF expression showed significantly higher mean MVD compared with those having negative VEGF expression (37.39, 95% CI 32.66-42.12 vs. 16.86, 95%CI 13.59-20.12 respectively, P < 0.001). Increasing mean MVD and VEGF positivity were significantly observed with higher grades of inflammation (P < 0.05). This work confirms a role for angiogenesis and increased VEGF expression in cutaneous LP and a relation of these events with the degree of inflammation in the disease.

Immunohistochemical expression of survivin in breast carcinoma: relationship with clinicopathological parameters, proliferation and molecular classification.

BACKGROUND AND OBJECTIVE: Survivin is a novel member of the inhibitor of apoptosis (IAP) gene family. It is associated with more aggressive behavior and parameters of poor prognosis in most human cancers including gastric, colorectal and bladder carcinomas. However, conflicting data exist on its prognostic effect in breast cancer. This current study is designed to assess survivin expression in breast carcinoma relating results with clinicopathological parameters, proliferation (MIB-1) and molecular classification MATERIAL AND METHODS: Our retrospective study comprised of 65 archived cases of breast carcinoma. Samples from the tumor and the adjacent normal breast tissue were immunostained for survivin and MIB-1. Nuclear and cytoplasmic survivin expression was evaluated in normal breast tissue and carcinoma regarding both the intensity and the percentage of positive cells. ER, PR, HER2 were used as surrogate markers to classify the cases into four molecular subtypes. RESULTS: Survivin expression was detected in 78.5% of breast carcinomas. The adjacent normal breast tissue was immunonegative. Survivin expression showed significant association with increased tumor size (p<0.0001), high histologic grade (p=0.04), lymph node metastases (p<0.001), advanced tumor stage (p<0.0001), MIB-1 expression (p=0.02), negative estrogen receptor status (p=0.01) and negative progesterone receptor status (p<0.0001). The subcellular localization of survivin significantly related to histologic grade, stage and lymph node involvement. The percentage of TNP (triple negative phenotype) and HER2+/ER-PR- tumors expressing survivin were significantly higher compared to the Luminal subtypes (p=0.01). CONCLUSION: Survivin expression was associated with parameters of poor prognosis in breast cancer. Moreover, the cancer-specific expression of survivin, coupled with its importance in inhibiting cell death and in regulating cell division, makes it a potential target for novel cancer treatment. KEY WORDS: Breast carcinoma - Immunohistochemistry - MIB-1 - Molecular classification - Survivin.