PDF - 419K, Activation of Kras concomitant with PTEN loss (PTENKO/Kras) results in endometrial tumors that invade the kidney parenchyma.

PDF - 306K, Over-expression of the human progesterone receptor (hPR) with a lentivirus in the tumor stroma.

PDF - 364K, Stromal or epithelial specific deletion of PR-A and PR-B.

PDF - 130K, Analysis of epithelial and stromal populations from PTENKO and PTENKO/Kras tumors.

PDF - 188K, Biologic behavior of PTENKO tumors during and after cessation of progesterone therapy.

PDF - 183K, Measurement of proliferative index and ER expression in PTENKO tumors treated under different hormonal conditions.

PDF - 250K, Histology of PTENKO tumors and hormone supplementation in treated mice.

PDF - 155K, Supplementary Table 1: Antibodies used for FACS sorting. Supplementary Table 2: Antibodies used for IHC. Supplementary Table 3: Primers used for QPCR. Supplementary Table 4: Antibodies used for Western blotting.Supplementary Table 5: Primers used for Methylation Specific. PCR

Cardiac ischemia-reperfusion (IR) leads to myocardial dysfunction by increasing production of reactive oxygen species (ROS). Mitochondrial H + leak decreases ROS formation; it has been postulated that increasing H + leak may be a mechanism of decreasing ROS production after IR. Ischemic preconditioning (IPC) decreases ROS formation after IR, but the mechanism is unknown. We hypothesize that pharmacologically increasing mitochondrial H + leak would decrease ROS production after IR. We further hypothesize that IPC would be associated with an increase in the rate of H + leak. Isolated male Sprague-Dawley rat hearts were subjected to either control or IPC. Mitochondria were isolated at end equilibration, end ischemia, and end reperfusion. Mitochondrial membrane potential (mΔΨ) was measured using a tetraphenylphosphonium electrode. Mitochondrial uncoupling was achieved by adding increasing concentrations of FCCP. Mitochondrial ROS production was measured by fluorometry using Amplex-Red. Pyridine dinucleotide levels were measured using HPLC. Before IR, increasing H + leak decreased mitochondrial ROS production. After IR, ROS production was not affected by increasing H + leak. H + leak increased at end ischemia in control mitochondria. IPC mitochondria showed no change in the rate of H + leak throughout IR. NADPH levels decreased after IR in both IPC and control mitochondria while NADH increased. Pharmacologically, increasing H + leak is not a method of decreasing ROS production after IR. Replenishing the NADPH pool may be a means of scavenging the excess ROS thereby attenuating oxidative damage after IR.

Abstract Progesterone, an agonist for the progesterone receptor (PR), can be an efficacious and well-tolerated treatment in endometrial cancer. The clinical use of progesterone is limited because of the lack of biomarkers that predict hormone sensitivity. Despite its efficacy in cancer therapy, mechanisms and site of action for progesterone remain unknown. Using an in vivo endometrial cancer mouse model driven by clinically relevant genetic changes but dichotomous responses to hormonal therapy, we show that signaling through stromal PR is necessary and sufficient for progesterone antitumor effects. Endometrial cancers resulting from epithelial loss of PTEN (PTENKO) were hormone sensitive and had abundant expression of stromal PR. Stromal deletion of PR as a single genetic change in these tumors induced progesterone resistance indicating that paracrine signaling through the stroma is essential for the progesterone therapeutic effects. A hormone-refractory endometrial tumor with low levels of stromal PR developed when activation of KRAS was coupled with PTEN-loss (PTENKO/Kras). The innate progesterone resistance in PTENKO/Kras tumors stemmed from methylation of PR in the tumor microenvironment. Add-back of stromal PR expressed from a constitutively active promoter sensitized these tumors to progesterone therapy. Results show that signaling through stromal PR is sufficient for inducing hormone responsiveness. Our findings suggest that epigenetic derepression of stromal PR could be a potential therapeutic target for sensitizing hormone-refractory endometrial tumors to progesterone therapy. On the basis of these results, stromal expression of PR may emerge as a reliable biomarker in predicting response to hormonal therapy. Cancer Res; 73(15); 4697–710. ©2013 AACR.

Ye, Weiping PhD, MD, MPH; Lee, Daniel BA; Ralston, James BA; Zweier, Jay MD; Crestanello, Juan MD Author Information

Brain natriuretic peptide (BNP) is increased after myocardial infarction and has prognostic implications. BNP may originate in the infarcted myocardium or in the non-infarcted myocardium secondary to changes in wall stress and loading conditions. We studied BNP expression in infarcted and non-

Hyponatremia (Na < 135mEq/L) is the most common electrolyte disorder in clinical practice. Hyponatremia increases mortality in patients with heart disease; however, the mechanisms remain unclear. W...

Background: Mitochondrial superoxide radical (O-2(center dot)) production increases after cardiac ischemia/reperfusion (IR). Ischemic preconditioning (IPC) preserves mitochondrial function and attenuates O-2(center dot) production, but the mechanism is unknown. Mitochondrial membrane potential (m Delta psi) is known to affect O-2(center dot) production; mitochondrial depolarization decreases O-2(center dot) formation. We examined the relationship between O-2(center dot) production and m Delta psi during IR and IPC.Materials/methods: Rat hearts were subjected to Control or IPC. Mitochondria were isolated at end equilibration (End EQ), end ischemia (End I), and end reperfusion (End RP). m Delta psi was measured using a tetraphenylphosphonium electrode. Mitochondrial O-2(center dot) production was measured by electron paramagnetic resonance using DMPO spin trap. Cytochrome c levels were measured using high- pressure liquid chromatography.Results: IPC preserved m Delta psi at End I (-156 +/- 5 versus -131 +/- 6 mV, P < 0.001) and End RP (-168 +/- 2 versus -155 +/- 2 mV, P < 0.05). At End RP, IPC attenuated O-2(center dot) production (2527 +/- 221 versus 3523 +/- 250 AU/mg protein, P < 0.05). IPC preserved cytochrome c levels (351 +/- 14 versus 269 +/- 16 picomoles/mg protein, P < 0.05) at End RP, and decreased mitochondrial cristae disruption (10% +/- 4% versus 33% +/- 7%, P < 0.05) and amorphous density formation (18% +/- 4% versus 28% +/- 1%, P < 0.05).Conclusion: We conclude that IPC preserves m Delta psi, possibly by limiting disruption of mitochondrial inner membrane. IPC also decreases mitochondrial O-2(center dot) production and preserves mitochondrial ultrastructure after IR. While it was previously held that slight decreases in m Delta psi decrease O-2(center dot) production, our results indicate that preservation of m Delta psi is associated with decreased O-2(center dot) and preservation of cardiac function in IPC. These findings indicate that the mechanism of IPC may not involve m Delta psi depolarization, but rather preservation of mitochondrial electrochemical potential. (C) 2012 Elsevier Inc. All rights reserved.

Hyponatremia (HN) is a condition in which the sodium concentration in the blood falls below the normal range disrupting the homeostasis of regular physiologic cell functions. This condition is common to patients with heart failure and often leads to mortality. In order to study the exact cause and effect of HN, an investigation with a rat model would be most ideal. In this study, we sought to evaluate the effect of HN by lowering NaCl and observe the outcome of correcting the osmolarity by increasing glucose in HN in isolated rat hearts.

Quarrie, Ricardo MD; Lee, Daniel S. BSc; Cramer, Brandon; Erdahl, Warren PhD; Pfeiffer, Douglas R. PhD; Zweier, Jay L. MD; Crestanello, Juan A. MD Author Information

Background. Proton leak (H+ leak) dissipates mitochondrial membrane potential (m Delta Psi) through the re-entry of protons into the mitochondrial matrix independent of ATP synthase. Changes in H+ leak may affect reactive oxygen species (ROS) production. We measured H+ leak and ROS production during ischemia-reperfusion and ischemic preconditioning (IPC) and examined how changing mitochondrial respiration affected m Delta Psi and ROS production.Materials and Methods. Isolated rat hearts (n = 6/group) were subjected to either control-IR or IPC. Rate pressure product (RPP) was measured. Mitochondria were isolated at end reperfusion. Respiration was measured by polarography and titrated with increasing concentrations of malonate (0.5-2 mM). m Delta Psi was measured using a tetraphenylphosphonium electrode. H+ leak is the respiratory rate required to maintain membrane potential at -150 mV in the presence of oligomycin-A. Mitochondrial complex III ROS production was measured by fluorometry using Amplex-red.Results. IPC improved recovery of RPP at end reperfusion (63% +/- 4% versus 21% +/- 2% in control-IR, P < 0.05). Ischemia-reperfusion caused increased H+ leak (94 +/- 12 versus 31 +/- 1 nmol O/mg protein/min in non-ischemic control, P < 0.05). IPC attenuates these increases (55 +/- 9 nmol O/mg protein/min, P < 0.05 versus control-IR). IPC reduced mitochondrial ROS production compared with control-IR (31 +/- 2 versus 40 +/- 3 nmol/mg protein/min, P < 0.05). As mitochondrial respiration decreased, m Delta Psi and mitochondrial ROS production also decreased. ROS production remained lower in IPC than in control-IR for all m Delta Psi and respiration rates.Conclusions. Increasing H+ leak is not associated with decreased ROS production. IPC decreases both the magnitude of H+ leak and ROS production after ischemia-reperfusion. (C) 2011 Elsevier Inc. All rights reserved.

Introduction: Mitochondrial reactive oxygen species (mROS) cause significant damage to myocardium after an episode of ischemia-reperfusion (IR). Ischemic preconditioning (IPC) decreases mROS formation and protects myocardium from IR. The mechanism of decreased mROS in IPC is unknown. Proton leak (H+ leak) dissipates mitochondria membrane potential (mΔΨ) and may regulate mROS production by changing the redox status of mitochondria. We hypothesize that increased H+ leak is responsible for decreased mROS production during IPC. Methods: Rat hearts were assigned to CONTROL (30 min of equilibration (EQ), 30 min ischemia (I), and 30 min of reperfusion (RP)) or IPC (10 min EQ, two 5 min episodes of IR, 30 min I, and 30 min RP). Rate pressure product (RPP) was measured throughout. Mitochondria were isolated at end equilibration (end EQ), end ischemia (end I), and end reperfusion (end RP). Respiration was measured by polarography, and mΔΨ with a TPP electrode. H+ leak is the respiratory rate required to maintain mΔΨ of -130mV in the presence of oligomycin-A, and was titrated with FCCP (0-160nM). Complex III mROS production was measured by fluorometry using Amplex-Red at end EQ and by electron paramagnetic resonance at end I and end RP. Results: Induction of IPC increased mROS production at end EQ (59±2 vs. 43±2 nmoles H2O2/mg protein/min, p<0.05, Fig.1) but did not change H+ leak (37±9 vs. 30±10 nmoles O/mg protein/min) when compared to CONTROL. IPC attenuated the increase in H+ leak at end I seen in CONTROL (44±11 vs. 140±11 nmoles O/mg protein/min, p<0.001). At end RP, IPC decreased mROS production (2115±146 vs. 3277±333 arbitrary units/mg protein, p<0.05). There was no difference in H+ leak between IPC and CONTROL at end RP (34±9 vs. 45±9 nmoles O/mg protein/min). IPC mitochondria responded to depolarization with greater reduction in mROS production than CONTROL (Fig.1). Further depolarization led to a trend of increased mROS in both CONTROL and IPC. The inflection point occurred at a higher mΔΨ in IPC mitochondria (-160 vs. -138 mV; Fig. 1). Conclusions: Increased rate of H+ leak is not the mechanism of decreased mROS after IR. While IPC did not lead to increased H+ leak, the induction of IPC increases the sensitivity of mitochondria to changes in mΔΨ leading to greater reduction in mROS per unit of change in mΔΨ. This increased sensitivity explains the greater reduction in mROS at end RP despite a lower H+ leak. Reducing mROS formation after ischemia-reperfusion is important to improving outcomes in patients with acute coronary syndrome.

Acute blockage of coronary artery results in myocardial injury manifested in clinical situations such as myocardial infarction (MI). Following ischemia, inflammation is provoked and may be related to the clinical outcomes. We studied the acute inflammatory response immediately following myocardial ischemia in human. Our results showed that in acute MI patients circulation levels of complement factor MASP-2 were significantly reduced comparing with those of healthy individuals or of coronary artery disease (CAD) patients without acute MI. This reduction was not due to genetic polymorphism of MASP-2 in acute MI patients. We hypothesized that MASP-2 was activated after acute myocardial ischemia, and tested this in cardiac patients undergoing surgically induced transient global heart ischemia. Our results showed that MASP-2 was significantly reduced in coronary circulation after global heart ischemia and was independently correlated with the post-operative increase of the myocardial injury marker, cardiac Troponin I. These data suggested that MASP-2 is activated in human acute heart ischemia and associated with myocardial injury.

Mitochondrial (mito) respiratory electron transfer chain (RTC) is a major source of reactive oxygen species (ROS) during ischemia reperfusion (IR). Mito membrane potential (Δψ) may affect ROS production by changing the redox status of the RTC. Mild uncoupling can lead to oxidation of the RTC and decrease ROS production. It is unclear how ischemic preconditioning (IPC) decreases mito ROS generation. We examined the changes in mito Δψ and its correlation to ROS production in IR and IPC mito.Isolated rat hearts (n=6/group) were subjected to 1) CONTROL: 30 minutes (min) of equilibration (EQ), 30 min of ischemia (I), and 30 min of reperfusion (RP), or 2) IPC: 10 min of EQ, two 5 min episodes of I and RP, 30 min of I, and 30 min of RP. Mitos were isolated at end I and end RP. ROS production was assessed by EPR spectroscopy using DMPO as spin trap. Δψ was measured using a TPP electrode. Data is expressed as mean±sem. ROS production by Complex I & III (arbitrary units/mg protein) Δψ (‐mV) End I End RP End I End RP CONTROL 2220±22 3524±340 121±8 150±2 IPC 1878±175 2479±296* 147±7* 160±3* p<0.05 vs. CONTROL Ischemia led to lower Δψ in IPC and CONTROL. Δψ recovered during reperfusion. However, Δψ remained higher in IPC at end I and end RP. IPC decreases ROS production during RP. Membrane depolarization was not associated with lower ROS production. We conclude that membrane depolarization is not responsible for the cardioprotective effect of IPC.