Deletion Of Protein Tyrosine Phosphatase 1B Research Articles

Endothelial PTP1B Deletion Promotes VWF Exocytosis and Venous Thromboinflammation.

Endothelial activation promotes the release of procoagulant extracellular vesicles and inflammatory mediators from specialized storage granules. Endothelial membrane exocytosis is controlled by phosphorylation. We hypothesized that the absence of PTP1B (protein tyrosine phosphatase 1B) in endothelial cells promotes venous thromboinflammation by triggering endothelial membrane fusion and exocytosis. Mice with inducible endothelial deletion of PTP1B (End.PTP1B-KO) underwent inferior vena cava ligation to induce stenosis and venous thrombosis. Primary endothelial cells from transgenic mice and human umbilical vein endothelial cells were used for mechanistic studies. Vascular ultrasound and histology showed significantly larger venous thrombi containing higher numbers of Ly6G (lymphocyte antigen 6 family member G)-positive neutrophils in mice with endothelial PTP1B deletion, and intravital microscopy confirmed the more pronounced neutrophil recruitment following inferior vena cava ligation. RT2 PCR profiler array and immunocytochemistry analysis revealed increased endothelial activation and adhesion molecule expression in primary End.PTP1B-KO endothelial cells, including CD62P (P-selectin) and VWF (von Willebrand factor). Pretreatment with the NF-κB (nuclear factor kappa B) kinase inhibitor BAY11-7082, antibodies neutralizing CD162 (P-selectin glycoprotein ligand-1) or VWF, or arginylglycylaspartic acid integrin-blocking peptides abolished the neutrophil adhesion to End.PTP1B-KO endothelial cells in vitro. Circulating levels of annexin V+ procoagulant endothelial CD62E+ (E-selectin) and neutrophil (Ly6G+) extracellular vesicles were also elevated in End.PTP1B-KO mice after inferior vena cava ligation. Higher plasma MPO (myeloperoxidase) and Cit-H3 (citrullinated histone-3) levels and neutrophil elastase activity indicated neutrophil activation and extracellular trap formation. Infusion of End.PTP1B-KO extracellular vesicles into C57BL/6J wild-type mice most prominently enhanced the recruitment of endogenous neutrophils, and this response was blunted in VWF-deficient mice or by VWF-blocking antibodies. Reduced PTP1B binding and tyrosine dephosphorylation of SNAP23 (synaptosome-associated protein 23) resulting in increased VWF exocytosis and neutrophil adhesion were identified as mechanisms, all of which could be restored by NF-κB kinase inhibition using BAY11-7082. Our findings show that endothelial PTP1B deletion promotes venous thromboinflammation by enhancing SNAP23 phosphorylation, endothelial VWF exocytosis, and neutrophil recruitment.

Abstract P3058: Cardiac-specific Deletion Of Ptp1b Induces A Global Metabolic And Lean Phenotype

About 13% of US adults have diabetes, and more than half of them exhibitcardiac hypertrophy and fibrosis, resulting in cardiac diastolic dysfunction,systolic dysfunction and heart failure. Obesity is the leading risk factor for type2 diabetes. The Global Burden of Disease 2019 listed the high BMI as the fifthrisk of preterm deaths for females, and the sixth for males worldwide. Thecurrent treatments for these diseases are ineffective providing an urgent needof new therapeutic targets. Protein tyrosine phosphatase 1B (PTP1B) is themaster regulator of insulin signaling and its expression in brain, heart, liver andadipose tissues are elevated in obese mice and humans. It has been reportedthat neuronal deletion of PTP1B decreased food intake, increased energyexpenditure and protected mice from diet-induced obesity. However, whetherPTP1B in peripheral tissues would also contribute to diet-induced obesityremains unclear. To test if cardiac-PTP1B plays roles in the development ofobesity, we generated cardiomyocyte-specific PTP1B knockout mice (PTP1BCM-KO mice) and fed them with high-fat diets. Our preliminary datademonstrated that, compared to control mice, the PTP1B CM-KO mice hadreduced body weight and improved glucose homeostasis. Further analysisrevealed that deletion of PTP1B in cardiomyocytes increased energyexpenditure but didn’t affect the food intake. Metabolomics data suggested fattyacid oxidation is elevated in PTP1B CM-KO mice heart, whereas the glycolysispathway is suppressed, the amount of AMP (Adenosine Monophosphate) inPTP1B CM-KO mice is elevated 86 times than control mice on HFD. Thesefindings were further verified by qPCR analysis and ELISA. PTP1B knockoutupregulated not only NAD+ (nicotinamide adenine dinucleotide) but alsoNADPH in the heart. Nicotinamide phosphoribosyltransferase (Nampt) is therate-limiting enzyme of the NAD + salvage pathway and exists in an intracellular(iNampt) and an extracellular (eNampt) form. iNampt plays a regulatory role inNAD + biosynthesis and affects energy homeostasis. eNampt has been reportedto act as a cytokine and associated with obesity and diabetes. In the PTP1BCM-KO mice, we observed a higher level of both iNampt and eNampt,suggesting a mechanism through iNampt locally and/or through eNamptsystematically. Based on these observations, we hypothesize that the deficiency of PTP1B in cardiomyocytes increases energy expenditure,protects mice from diet-induced obesity and attenuates cardiacmalfunction induced by obesity, and cardiac-PTP1B governs whole bodyenergy homeostasis through Nampt and NAD + . We will test our hypothesisin the following specific Aims: Aim1: To elucidate the role of PTP1B in cardiac function and glucosehomeostasis.Aim2: To determine if and how the deletion of cardiac-PTP1B affectenergy balance and metabolic status through iNampt locally and/oreNampt systemically.

Computational analysis of PTP-1B site-directed mutations and their structural binding to potential inhibitors

Protein tyrosine phosphatase-1B (PTP-1B) is a well-known therapeutic target for diabetes and obesity as it suppresses insulin and leptin signaling. PTP-1B deletion or pharmacological suppression boosted glucose homeostasis and insulin signaling without altering hepatic fat storage. Inhibitors of PTP-1B may be useful in the treatment of type 2 diabetes, and shikonin, a naturally occurring naphthoquinone dye pigment, is reported to inhibit PTP-1B and possess antidiabetic properties. Since the cell contains a large number of phosphatases, PTP-1B inhibitors must be effective and selective. To explore more about the mechanism underlying the inhibitor's efficacy and selectivity, we investigated its top four pharmacophores and used site-directed mutagenesis to insert amino acid mutations into PTP-1B as an extension of our previous study where we identified 4 pharmacophores of shikonin. The study aimed to examine the site-directed mutations like R24Y, S215E, and S216C influence the binding of shikonin pharmacophores, which act as selective inhibitors of PTP-1B. To achieve this purpose, docking and molecular dynamics simulations of wild-type (WT) and mutant PTP-1B with antidiabetic compounds were undertaken. The simulation results revealed that site-directed mutations can change the hydrogen bond and hydrophobic interactions between shikonin pharmacophores and many residues in PTP-1B's active site, influencing the drug's binding affinity. These findings could aid researchers in better understanding PTP-1B inhibitors' selective binding mechanism and pave the path for the creation of effective PTP-1B inhibitors.

Engineering iPSC-derived neutrophils to fight infection

Abstract Neutrophils are the initial responders to infection and injury and are critical for host survival. Immunocompromised patients are prone to recurrent bacterial and fungal infections which may require granulocyte transfusion. However, these transfusions have minimal efficacy as donor material is limited and highly heterogeneous. Induced pluripotent stem cells (iPSCs) are a promising option for generating a defined therapeutic that can improve patient outcomes. Therefore, we aimed to engineer iPSC-derived neutrophils to improve recruitment and antimicrobial function. Prior research in the field has shown that iPSC-derived neutrophils have limited function compared to primary human neutrophils, likely due to inhibitory signaling pathways. To generate iPSC-derived neutrophils with enhanced capacity, we utilized CRISPR/Cas9 to delete the protein tyrosine phosphatase 1B (PTP1B), a negative regulator of neutrophil function. PTP1B−/− neutrophils displayed increased cellular migration and inflammatory cytokine signaling in response to bacterial stimuli versus WT cells. Additionally, upon selection of cells with mature neutrophil markers, these PTP1B−/− neutrophils show similar fungal killing ability and improved phagocytosis compared to WT. Thus, these data indicate that deletion of PTP1B can improve the antimicrobial function of iPSC neutrophils and highlights the value of our system for genetically engineering iPSC-derived neutrophils. Supported by grants from the NIH (5R01AI134749-03 and Microbes in Health and Disease T32 NIAID: T32AI055397)

Protein tyrosine phosphatase 1B regulates miR-208b-argonaute 2 association and thyroid hormone responsiveness in cardiac hypertrophy.

Increased production of reactive oxygen species plays an essential role in the pathogenesis of several diseases, including cardiac hypertrophy. In our search to identify redox-sensitive targets that contribute to redox signaling, we found that protein tyrosine phosphatase 1B (PTP1B) was reversibly oxidized and inactivated in hearts undergoing hypertrophy. Cardiomyocyte-specific deletion of PTP1B in mice (PTP1B cKO mice) caused a hypertrophic phenotype that was exacerbated by pressure overload. Furthermore, we showed that argonaute 2 (AGO2), a key component of the RNA-induced silencing complex, was a substrate of PTP1B in cardiomyocytes and in the heart. Our results revealed that phosphorylation at Tyr393 and inactivation of AGO2 in PTP1B cKO mice prevented miR-208b-mediated repression of thyroid hormone receptor-associated protein 1 (THRAP1; also known as MED13) and contributed to thyroid hormone-mediated cardiac hypertrophy. In support of this conclusion, inhibiting the synthesis of triiodothyronine (T3) with propylthiouracil rescued pressure overload-induced hypertrophy and improved myocardial contractility and systolic function in PTP1B cKO mice. Together, our data illustrate that PTP1B activity is cardioprotective and that redox signaling is linked to thyroid hormone responsiveness and microRNA-mediated gene silencing in pathological hypertrophy.

Deletion of PTP1B From Brain Neurons Partly Protects Mice From Diet-Induced Obesity and Minimally Improves Fertility.

Reproductive dysfunction in women has been linked to high caloric diet (HCD)-feeding and obesity. Central resistance to leptin and insulin have been shown to accompany diet-induced infertility in rodent studies, and we have previously shown that deleting suppressor of cytokine signaling 3, which is a negative regulator of leptin signaling, from all forebrain neurons partially protects mice from HCD-induced infertility. In this study, we were interested in exploring the role of protein tyrosine phosphatase 1B (PTP1B), which is a negative regulator of both leptin and insulin signaling, in the pathophysiology of HCD-induced obesity and infertility. To this end, we generated male and female neuron-specific PTP1B knockout mice and compared their body weight gain, food intake, glucose tolerance, and fertility relative to control littermates under both normal calorie diet and HCD feeding conditions. Both male and female mice with neuronal PTP1B deletion exhibited slower body weight gain in response to HCD feeding, yet only male knockout mice exhibited improved glucose tolerance compared with controls. Neuronal PTP1B deletion improved the time to first litter in HCD-fed mice but did not protect female mice from eventual HCD-induced infertility. While the mice fed a normal caloric diet remained fertile throughout the 150-day period of assessment, HCD-fed females became infertile after producing only a single litter, regardless of their genotype. These data show that neuronal PTP1B deletion is able to partially protect mice from HCD-induced obesity but is not a critical mediator of HCD-induced infertility.

Selective deficiency in endothelial PTP1B protects from diabetes and endoplasmic reticulum stress-associated endothelial dysfunction via preventing endothelial cell apoptosis

Diabetes notably increases the risk for endothelial dysfunction, a main precursor for microvascular complications. While endoplasmic reticulum stress (ERS) and protein tyrosine phosphatase 1B (PTP1B) have been associated with endothelial dysfunction in resistance vessels, whether these mechanisms also contribute to diabetes-mediated endothelial dysfunction in conduit arteries remains unknown. Herein, we tested the hypothesis that diabetes induces macrovascular endothelial dysfunction via endothelial ERS-induced, PTP1B-mediated apoptosis. We showed that diabetes concomitantly increased the expression of PTP1B and of markers of ERS, including GRP78, XBP1, splXBP1 and CHOP in human vessels. Exposure of aortic rings from wild-type mice to the ERS inducers tunicamycin and thapsigargin markedly reduced endothelium-dependent relaxation. Global and endothelial-specific deletion of PTP1B as well as pharmacological inhibition protected aortic rings from ERS-mediated endothelial dysfunction. Nitric oxide synthase inhibition with l-NAME abolished relaxation in the presence and absence of ERS, but neither reactive oxygen species scavenging with tempol or peg-catalase, nor cyclooxygenase inhibition with indomethacin prevented ERS-mediated endothelial dysfunction. However, both p38-MAPK and JNK inhibition protected aortic rings from ERS-mediated endothelial dysfunction. In HUVECs, PTP1B deletion prevented ERS-induced PARP cleavage and apoptosis. Lastly, acute ERS inhibition in aortic rings and selective deficiency of endothelial PTP1B in mice protected mice from diabetes-induced endothelial dysfunction. Altogether, these data support the contribution of the p38/JNK-apoptosis pathway in ERS-mediated endothelial dysfunction and present endothelial PTP1B as a major regulator of endothelial cell viability in conduit vessels and a potential target for the management of macrovascular diseases in diabetes.

Protein tyrosine phosphatase 1b deficiency protects against hepatic fibrosis by modulating nadph oxidases

Inflammation is typically associated with the development of fibrosis, cirrhosis and hepatocellular carcinoma. The key role of protein tyrosine phosphatase 1B (PTP1B) in inflammatory responses has focused this study in understanding its implication in liver fibrosis. Here we show that hepatic PTP1B mRNA expression increased after bile duct ligation (BDL), while BDL-induced liver fibrosis was markedly reduced in mice lacking Ptpn1 (PTP1B−/−) as assessed by decreased collagen deposition and α-smooth muscle actin (α-SMA) expression. PTP1B−/− mice also showed a significant increase in mRNA levels of key markers of monocytes recruitment (Cd68, Adgre1 and Ccl2) compared to their wild-type (PTP1B+/+) littermates at early stages of injury after BDL. Interestingly, the lack of PTP1B strongly increased the NADPH oxidase (NOX) subunits Nox1/Nox4 ratio and downregulated Cybb expression after BDL, revealing a pro-survival pattern of NADPH oxidase induction in response to liver injury. Chimeric mice generated by transplantation of PTP1B−/− bone marrow (BM) into irradiated PTP1B+/+ mice revealed similar hepatic expression profile of NOX subunits than PTP1B−/− mice while these animals did not show differences in infiltration of myeloid cells at 7 days post-BDL, suggesting that PTP1B deletion in other liver cells is necessary for boosting the early inflammatory response to the BDL. PTP1B−/− BM transplantation into PTP1B+/+ mice also led to a blockade of TGF-β and α-SMA induction after BDL. In vitro experiments demonstrated that deficiency of PTP1B in hepatocytes protects against bile acid-induced apoptosis and abrogates hepatic stellate cells (HSC) activation, an effect ameliorated by NOX1 inhibition. In conclusion, our results have revealed that the lack of PTP1B switches NOX expression pattern in response to liver injury after BDL and reduces HSC activation and liver fibrosis.

Inducible Knockdown of Endothelial Protein Tyrosine Phosphatase-1B Promotes Neointima Formation in Obese Mice by Enhancing Endothelial Senescence.

Protein tyrosine phosphatase-1B (PTP1B) is a negative regulator of receptor tyrosine kinase signaling. In this study, we determined the importance of PTP1B expressed in endothelial cells for the vascular response to arterial injury in obesity. Morphometric analysis of vascular lesions generated by 10% ferric chloride (FeCl3) revealed that tamoxifen-inducible endothelial PTP1B deletion (Tie2.ERT2-Cre × PTP1Bfl/fl; End.PTP1B knockout, KO) significantly increased neointima formation, and reduced numbers of (endothelial lectin-positive) luminal cells in End.PTP1B-KO mice suggested impaired lesion re-endothelialization. Significantly higher numbers of proliferating cell nuclear antigen (PCNA)-positive proliferating cells as well as smooth muscle actin (SMA)-positive or vascular cell adhesion molecule-1 (VCAM1)-positive activated smooth muscle cells or vimentin-positive myofibroblasts were detected in neointimal lesions of End.PTP1B-KO mice, whereas F4/80-positive macrophage numbers did not differ. Activated receptor tyrosine kinase and transforming growth factor-beta (TGFβ) signaling and oxidative stress markers were also significantly more abundant in End.PTP1B-KO mouse lesions. Genetic knockdown or pharmacological inhibition of PTP1B in endothelial cells resulted in increased expression of caveolin-1 and oxidative stress, and distinct morphological changes, elevated numbers of senescence-associated β-galactosidase-positive cells, and increased expression of tumor suppressor protein 53 (p53) or the cell cycle inhibitor cyclin-dependent kinase inhibitor-2A (p16INK4A) suggested senescence, all of which could be attenuated by small interfering RNA (siRNA)-mediated downregulation of caveolin-1. In vitro, senescence could be prevented and impaired re-endothelialization restored by preincubation with the antioxidant Trolox. Our results reveal a previously unknown role of PTP1B in endothelial cells and provide mechanistic insights how PTP1B deletion or inhibition may promote endothelial senescence. Absence of PTP1B in endothelial cells impairs re-endothelialization, and the failure to induce smooth muscle cell quiescence or to protect from circulating growth factors may result in neointimal hyperplasia.

Reduced Insulin Resistance Contributes to the Beneficial Effect of Protein Tyrosine Phosphatase-1B Deletion in a Mouse Model of Sepsis.

Hyperglycemia is a common feature of septic patients and has been associated with poor outcome and high mortality. In contrast, insulin has been shown to decrease mortality and to prevent the incidence of multiorgan failure but is often associated with deleterious hypoglycemia. Protein Tyrosine Phosphatase 1B (PTP1B) is a negative regulator of both insulin signaling and NO production, and has been shown to be an aggravating factor in septic shock. To evaluate the potential therapeutic effect of PTP1B blockade on glucose metabolism and insulin resistance in an experimental model of sepsis, we assessed the effect of PTP1B gene deletion in a cecal ligation and puncture (CLP) model of sepsis. PTP1B gene deletion significantly limited CLP-induced insulin resistance, improved AMP-activated protein kinase signaling pathway and Glucose Transporter 4 translocation, and decreased inflammation. These effects were associated with a reduction of sepsis-induced endothelial dysfunction/impaired NO production and especially of insulin-mediated dilatation. This modulation of insulin resistance may contribute to the beneficial effect of PTP1B blockade in septic shock, especially in terms of inflammation and cardiac metabolism.

Role of protein tyrosine phosphatase 1B in cardiovascular diseases

Protein Tyrosine Phosphatase 1B (PTP1B) is mostly involved in negative regulation of signaling mediated by Tyrosine Kinase Receptors, especially the insulin and leptin receptors. This enzyme thus plays a major role in the development of diseases associated with insulin resistance, such as obesity and diabetes. PTP1B inhibition is currently considered as an attractive treatment of insulin resistance and associated metabolic disorders. In parallel, emerging evidence also suggests that PTP1B is widely expressed in cardiovascular tissues, notably in the heart and the endothelium, and that it could also be a potential treatment of several cardiovascular diseases. PTP1B is especially present in endothelial cells, and appears to contribute to endothelial dysfunction. Indeed, preclinical evidence shows that pharmacological inhibition of gene deletion of PTP1B reduces endothelial dysfunction in various cardiovascular diseases associated or not with insulin resistance. In parallel, because PTP1B also negatively modulates VEGF signaling, inhibition of this enzyme also tends to favor cardiac angiogenesis. Importantly, blocking PTP1B also results in beneficial effects on cardiac dysfunction and remodeling not only in metabolic diseases but also in the context of heart failure, thus this enzyme represents an attractive new target for the treatment of this disease. This beneficial effect in heart failure may to a large extent result from the endothelial protective and/or proangiogenic effects of PTP1B blockade. Finally, PTP1B inhibition also reduces cardiac dysfunction, but also systemic inflammation and mortality in experimental models of septic shock, and thus may also constitute a new treatment of this disease. Altogether, accumulating preclinical evidence suggests that PTP1B represents an interesting molecular target to treat both cardiovascular and metabolic diseases, which often share the same risk factors. This concept now deserves to be tested in clinical studies that should soon be possible with the current development of selective PTP1B inhibitors.

Abstract 037: Role of Pro-opiomelanocortin (POMC) Specific PTP1B in Differential Regulation of Blood Pressure, Liver Lipid Accumulation and Glucose Tolerance in Response to a High Fat Diet

Obese subjects are often resistant to leptin’s metabolic effects although blood pressure (BP) and sympathetic nervous system responses appear to be preserved. Protein tyrosine phosphatase 1B (PTP1B), a negative regulator of leptin signaling, may play a role in promoting this selective leptin resistance and causing metabolic dysfunction in obesity. Our previous studies suggest that the chronic BP responses to leptin are mediated via activation of pro-opiomelanocortin (POMC) neurons. The goal of this study was to determine if PTP1B in POMC neurons differentially controls metabolic functions and BP in mice fed a high fat diet (HFD). Male mice with POMC specific PTP1B deletion (POMC/PTP1B -/- ) and littermate controls (PTP1B flox/flox ) were fed a HFD from 6 to 22 wks of age. Baseline BP after 16 weeks of a HFD (95±2 vs. 95±3 mmHg) and BP responses to acute stress (Δ32±0 vs. Δ32±6 mmHg), measured by telemetry, were not different in POMC/PTP1B -/- compared to control mice, respectively. Heart rate (HR) was not different in POMC/PTP1B -/- and control mice during acute stress (699±4 vs. 697±15 bpm, respectively). Total body weight (TBW) and fat mass were reduced at 20 weeks of age in POMC/PTP1B -/- compared to controls (36.7±0.1 vs. 42.0±1 g TBW and 12.7±0.4 vs. 16.1±1.0 g fat mass, respectively). Liver weight of POMC/PTP1B -/- mice was less than in controls, and this was evident even when liver weight was normalized as % of TBW (4.5±0.2 vs. 5.0±0.2 %). POMC/PTP1B -/- males had reduced liver lipid accumulation compared to controls as measured by EchoMRI (0.08±0.03 vs. 0.15±0.03 g/g liver weight). Glucose tolerance was also improved by 46% in POMC/PTP1B -/- compared to controls as measured by AUC, 25856±1683 vs. 47267±5616 mg/dLx120min, respectively. These findings indicate that PTP1B signaling in POMC neurons plays a crucial role in regulating liver lipid accumulation and glucose tolerance but does not appear to mediate changes in BP or BP responses to acute stress in mice fed a high HFD (supported by NHLBI-PO1HL51971 and NIGMS P20GM104357)

Endothelial deletion of protein tyrosine phosphatase-1B protects against pressure overload-induced heart failure in mice.

Cardiac angiogenesis is an important determinant of heart failure. We examined the hypothesis that protein tyrosine phosphatase (PTP)-1B, a negative regulator of vascular endothelial growth factor (VEGF) receptor-2 activation, is causally involved in the cardiac microvasculature rarefaction during hypertrophy and that deletion of PTP1B in endothelial cells prevents the development of heart failure. Cardiac hypertrophy was induced by transverse aortic constriction (TAC) in mice with endothelial-specific deletion of PTP1B (End.PTP1B-KO) and controls (End.PTP1B-WT). Survival up to 20 weeks after TAC was significantly improved in mice lacking endothelial PTP1B. Serial echocardiography revealed a better systolic pump function, less pronounced cardiac hypertrophy, and left ventricular dilation compared with End.PTP1B-WT controls. Histologically, banded hearts from End.PTP1B-KO mice exhibited increased numbers of PCNA-positive, proliferating endothelial cells resulting in preserved cardiac capillary density and improved perfusion as well as reduced hypoxia, apoptotic cell death, and fibrosis. Increased relative VEGFR2 and ERK1/2 phosphorylation and greater eNOS expression were present in the hearts of End.PTP1B-KO mice. The absence of PTP1B in endothelial cells also promoted neovascularization following peripheral ischaemia, and bone marrow transplantation excluded a major contribution of Tie2-positive haematopoietic cells to the improved angiogenesis in End.PTP1B-KO mice. Increased expression of caveolin-1 as well as reduced NADPH oxidase-4 expression, ROS generation and TGFβ signalling were observed and may have mediated the cardioprotective effects of endothelial PTP1B deletion. Endothelial PTP1B deletion improves cardiac VEGF signalling and angiogenesis and protects against chronic afterload-induced heart failure. PTP1B may represent a useful target to preserve cardiac function during hypertrophy.

Selective Vascular Endothelial Protection Reduces Cardiac Dysfunction in Chronic Heart Failure.

Chronic heart failure (CHF) induces endothelial dysfunction in part because of decreased nitric oxide (NO(·)) production, but the direct link between endothelial dysfunction and aggravation of CHF is not directly established. We previously reported that increased NO production via inhibition of protein tyrosine phosphatase 1B (PTP1B) is associated with reduced cardiac dysfunction in CHF. Investigation of the role of endothelial PTP1B in these effects may provide direct evidence of the link between endothelial dysfunction and CHF. Endothelial deletion of PTP1B was obtained by crossing LoxP-PTP1B with Tie2-Cre mice. CHF was assessed 4 months after myocardial infarction. In some experiments, to exclude gene extinction in hematopoietic cells, Tie2-Cre/LoxP-PTP1B mice were lethally irradiated and reconstituted with bone marrow from wild-type mice, to obtain mouse with endothelial-specific deletion of PTP1B. Vascular function evaluated ex vivo in mesenteric arteries showed that in wild-type mice, CHF markedly impaired NO-dependent flow-mediated dilatation. CHF-induced endothelial dysfunction was less marked in endoPTP1B(-/-) mice, suggesting restored NO production. Echocardiographic, hemodynamic, and histological evaluations demonstrated that the selectively improved endothelial function was associated with reduced left ventricular dysfunction and remodeling, as well as increased survival, in the absence of signs of stimulated angiogenesis or increased cardiac perfusion. Prevention of endothelial dysfunction, by endothelial PTP1B deficiency, is sufficient to reduce cardiac dysfunction post myocardial infarction. Our results provide for the first time a direct demonstration that endothelial protection per se reduces CHF and further suggest a causal role for endothelial dysfunction in CHF development.

Abstract 11602: Direct Evidence That Vascular Endothelial Protection Leads to aa Reduction of Heart Failure

Introduction: Heart failure (HF) induces endothelial dysfunction, and especially decreases nitric oxide (NO) production, but the direct link between endothelial dysfunction and aggravation of HF is unknown. We previously reported a new treatment of endothelial dysfunction, based on inhibition of protein tyrosine phosphatase 1B (PTP1B), which both increases NO production and reduces cardiac dysfunction in HF. However the exact role of endothelial PTP1B in these effects is unknown. Hypothesis: Specific endothelial deletion of PTP1B reduces HF by limiting endothelial dysfunction Methods: Endothelial deletion of PTP1B was obtained by crossing LoxP-PTP1B mice with mice expressing Cre recombinase under the control of the endothelial promoter Tie2. Since this Tie2-Cre approach is known to also induce gene extinction in hematopoietic cells, Tie2Cre(+)PTP1Bf/f mice were also lethally irradiated and reinjected with bone marrow from wild type (WT; CD45.1) mice, in order to obtain a specific deletion of endothelial PTP1B (endoPTP1B-/- mice). All mice were then subjected to 4 months of myocardial infarction (MI) in order to induce HF, or to sham surgery Results: Vascular function evaluated ex vivo in mesenteric arteries (arteriograph) showed that in WT mice, HF markedly impaired endothelium-dependent, flow-mediated dilatation (sham: 40±4%; HF: 5±5; p<0.001). This HF-induced endothelial dysfunction was less marked in Tie2Cre(+)PTP1Bf/f (MI: 30±5; p<0.001 vs. WT) and in endoPTP1B-/- mice (35±6%). This improved response in endoPTP1B-/- mice were abolished by a NO-synthase inhibitor, suggesting a restored NO production. Evaluation of cardiac function by echocardiography showed that in WT, MI decreased left ventricular fractional shortening (sham: 49±2; HF: 12±1%; p<0.001) and which was improved in Tie2Cre(+)PTP1Bf/f (26±1%; p<0.01 vs. WT), and in endoPTP1B-/- mice (29±1%; p<0.01 vs. WT). In parallel, endothelial PTP1B deficiency was associated with an increased survival. Conclusions: Endothelial PTP1B deficiency induced an improvement of endothelial function but especially improved cardiac function. These results provide a direct demonstration of the beneficial effect of endothelial protection in HF.

Deletion of Protein Tyrosine Phosphatase 1B (PTP1B) Enhances Endothelial Cyclooxygenase 2 Expression and Protects Mice from Type 1 Diabetes-Induced Endothelial Dysfunction.

Protein tyrosine phosphatase 1B (PTP1B) dephosphorylates receptors tyrosine kinase and acts as a molecular brake on insulin signaling pathway. Conditions of metabolic dysfunction increase PTP1B, when deletion of PTP1B protects against metabolic disorders by increasing insulin signaling. Although vascular insulin signaling contributes to the control of glucose disposal, little is known regarding the direct role of PTP1B in the control of endothelial function. We hypothesized that metabolic dysfunctions increase PTP1B expression in endothelial cells and that PTP1B deletion prevents endothelial dysfunction in situation of diminished insulin secretion. Type I diabetes (T1DM) was induced in wild-type (WT) and PTP1B-deficient mice (KO) with streptozotocin (STZ) injection. After 28 days of T1DM, KO mice exhibited a similar reduction in body weight and plasma insulin levels and a comparable increase in glycemia (WT: 384±20 vs. Ko: 432±29 mg/dL), cholesterol and triglycerides, as WT mice. T1DM increased PTP1B expression and impaired endothelial NO-dependent relaxation, in mouse aorta. PTP1B deletion did not affect baseline endothelial function, but preserved endothelium-dependent relaxation, in T1DM mice. NO synthase inhibition with L-NAME abolished endothelial relaxation in control and T1DM WT mice, whereas L-NAME and the cyclooxygenases inhibitor indomethacin were required to abolish endothelium relaxation in T1DM KO mice. PTP1B deletion increased COX-2 expression and PGI2 levels, in mouse aorta and plasma respectively, in T1DM mice. In parallel, simulation of diabetic conditions increased PTP1B expression and knockdown of PTP1B increased COX-2 but not COX-1 expression, in primary human aortic endothelial cells. Taken together these data indicate that deletion of PTP1B protected endothelial function by compensating the reduction in NO bioavailability by increasing COX-2-mediated release of the vasodilator prostanoid PGI2, in T1DM mice.

PTP1B Deletion Enhances Endothelial Cyclooxygenase 2 Expression and Protects Mice from Type 1 Diabetes‐Induced Endothelial Dysfunction

Protein tyrosine phosphatase 1B (PTP1B) dephosphorylates receptors tyrosine kinase and acts as a molecular brake on insulin signaling. Although vascular insulin signaling contributes to the control of glucose disposal, little is known regarding the role of PTP1B in the control of endothelial function. We hypothesized that metabolic dysfunctions increase PTP1B expression in endothelial cells and that PTP1B deletion prevents endothelial dysfunction in situation of diminished insulin secretion. Type I diabetes (T1DM) was induced in wild-type (WT) and PTP1B-deficient mice (KO) with streptozotocin (STZ). After 28 days of T1DM, body weight reduction, decrease in plasma insulin levels and increased in glycemia, cholesterol and triglycerides, were similar in WT and KO mice. T1DM increased PTP1B expression and impaired aortic endothelial NO-dependent relaxation. PTP1B deletion did not affect baseline endothelial NO-dependent relaxation, but preserved endothelium-dependent relaxation, in T1DM mice. NO synthase inhibition with L-NAME abolished endothelial relaxation in control and T1DM WT mice. L-NAME and the cyclooxygenases inhibitor indomethacin were required to abolish endothelium relaxation in T1DM KO mice. PTP1B deletion increased COX-2 expression and PGI2 levels, in mouse aorta and plasma respectively, in T1DM mice. In parallel, simulation of diabetic conditions increased PTP1B expression and knockdown of PTP1B increased COX-2 but not COX-1 expression, in primary human aortic endothelial cells. These data indicate that deletion of PTP1B protected endothelial function by compensating the reduction in NO bioavailability by increasing COX-2-mediated release of the vasodilator prostanoid PGI2, in T1DM mice.

Deletion of myeloid-PTP1B decreases MHC Class I expression and peptide presentation through an IL-10 dependent mechanism in response to LPS challenge

Protein Tyrosine Phosphatase 1B (PTP1B) inhibition is a target in the treatment of type 2 Diabetes Mellitus, and as such PTP1B inhibitors are in Phase II clinical trials. Previously our laboratory demonstrated that myeloid-specific deletion of PTP1B (LysM PTP1B) results in an increase in systemic IL-10 secretion and expression. In the current work we investigated how PTP1B deficiency affects the activation phenotype of murine macrophages in response to inflammatory stimuli. We demonstrate that myeloidspecific PTP1B deletion results in a decrease in expression of MHC Class I, along with co-stimulatory molecules CD80 and CD40. Interaction assays reveal a defect in the cells’ ability to activate reporter B3Z T cells. Myeloidspecific PTP1B deletion increases the percentage of bone-marrow-derived-macrophages (BMDMs) positive for IL-10 which is associated with a decrease in iNOS production. Western blotting analysis demonstrated hyperphosporylation of ERK1/2 which has been suggested before to improve access to the IL-10 promoter. This provides evidence to suggest that myeloid-PTP1B deletion decreases MHC Class I expression and peptide presentation through an IL-10-dependent mechanism. Published: 16 April 2015

Abstract 429: PTP1B, a Link Between Endoplasmic Reticulum Stress and Endothelial Dysfunction

Endothelial dysfunction is a common feature of diabetes and a major contributor to diabetic vascular complications. Despite aggressive diabetes management strategies, preventing diabetes-induced endothelial dysfunction remains a difficult challenge. Endothelium insulin resistance and endoplasmic reticulum (ER) stress have been reported to contribute to endothelial dysfunction. Protein tyrosine phosphatase 1B (PTP1B) is a phosphatase bound to the ER known to regulate insulin sensitivity and ER stress. We hypothesize that diabetes alters PTP1B regulation of endothelial cell function and that inhibiting PTP1B may allow a strategy to prevent diabetes-induced endothelial dysfunction. Using immuno-histochemistry we showed that PTP1B is expressed in the endothelial cells of human saphenous vein, and in human primary aortic endothelial cells. Western-blot and quantitative real-time PCR analysis revealed that diabetes increased PTP1B and ER stress markers expression (CHOP: 1.4±0.2, GRP78: 1.5±0.2, XBP1spliced: 2.1±0.3, XBP1: 1.4±0.2 fold changes), in human saphenous veins. Deletion of PTP1B in obese diabetic mice (db/db) significantly reduced the level of expression (mRNA) of ER stress markers in mouse aorta. Incubation of mouse aortic rings with tunicamycin (tunica) or thapsigargin (thapsi), two ER stress inducers, increased the level of expression of ER stress markers (mRNA) and significantly reduced endothelium-dependent relaxation. (Tunica: -17±5%, thapsi: -30±6%, vs. DMSO). NO synthase inhibition with LNAME completely abolished endothelium-dependent relaxation in vehicle and tunica-treated rings. Neither the reactive oxygen species (ROS) scavenger tempol, nor catalase restored endothelial function, in tunica-treated rings. This suggests that ER stress induces a reduction in NO bioavailability independent of ROS. Aortic rings from PTP1B KO mice were protected against ER stress-induced endothelial dysfunction and exhibited a modest increase in ER stress markers compared to control rings, in response to tunica. All together these data demonstrate that PTP1B deletion prevents ER stress-induced endothelial dysfunction and that targeting PTP1B might be a beneficial approach to restore diabetic endothelial dysfunction.

Complement-mediated glomerular injury is reduced by inhibition of protein-tyrosine phosphatase 1B.

The unfolded protein response and endoplasmic reticulum-associated degradation (ERAD) contribute to injury in renal glomerular diseases, including those mediated by complement C5b-9. In the present study, we address the role of protein-tyrosine phosphatase 1B (PTP1B) in complement-mediated glomerular injury and ERAD. In glomerular epithelial cells (GECs)/podocytes and PTP1B-deficient mouse embryonic fibroblasts exposed to complement, inhibition/deletion of PTP1B reduced ERAD, as monitored by the ERAD reporter CD3δ. Overexpression of PTP1B produced an effect similar to PTP1B deficiency on ERAD in complement-treated GECs. Complement-mediated cytotoxicity was reduced after PTP1B overexpression and tended to be reduced after PTP1B inhibition. PTP1B enhanced the induction of certain ERAD components via the inositol-requiring-1α branch of the unfolded protein response. PTP1B knockout mice with anti-glomerular basement membrane glomerulonephritis had decreased proteinuria and showed less podocyte loss and endoplasmic reticulum dysfunction compared with wild-type littermates. These results imply that endogenous levels of PTP1B are tightly regulated and that both overexpression and inhibition can affect ERAD. The cytoprotective effects of PTP1B deletion in cultured cells and in anti-glomerular basement membrane nephritis suggest that PTP1B may potentially be a therapeutic target in complement-mediated diseases.