Induction of RAGE-NFkB signalling axis enhances SHP-2 tyrosine phosphatase expression resulting in deviant activation of diabetic monocytes

Abstract Purpose Aberrant activation of Type 2 Diabetes mellitus (T2DM) monocytes is an important pathomechanism leading to restricted arteriogenesis and augmented atherosclerosis, hereby, accelerating CAD and PAD. Tyrosine phosphatase SHP-2 was found to be upregulated in T2DM-monocytes. This study aimed to identify the pathways regulating SHP-2 expression in T2DM-monocytes. Methods Primary human monocytes were isolated from the peripheral blood of T2DM patients and healthy individuals. Monocytes were incubated with Methylglyoxal (MG), a highly reactive side product of glycolysis, Receptor for advanced glycation end product (RAGE) ligand AGE-bovine serum (AGE-BSA) or TNFα for 24 hours. Transwell migration assays were used to analyse the migratory potential of monocytes. Western Blot, RT-qPCR and FACS were performed to quantify the expression of relevant molecules. Pharmacological inhibitors were used to study functional relevance of the RAGE-NFκB-SHP-2 signalling axis. Results Significantly enhanced SHP-2 expression was detected in monocytes, which were incubated with TNFα, MG or AGE-BSA, respectively. Co-incubation of these molecules with NFκB-inhibitor blocked SHP-2 upregulation. Pharmacological inhibition of RAGE reversed the MG or AGE-BSA induced SHP-2 expression and activity in monocytes. RAGE expression on monocytes was upregulated after the incubation with MG or AGE-BSA, consistent with enhanced RAGE mRNA levels in T2DM monocytes. Besides, we also detected elevated SHP-2 transcripts in monocytes of T2DM patients which was more pronounced in monocytes with augmented TNFα expression. Furthermore, MG and AGE-BSA provoked the enhanced migration of monocytes which could be significantly reduced after the application of an allosteric SHP-2 inhibitor. Interestingly, pharmacological inhibition of RAGE in these conditions alone was sufficient to block the elevated monocyte migration. Moreover, monocytes isolated from T2DM patients revealed a comparable pro-migratory phenotype, which was completely restored after the pharmacological inhibition of SHP-2. Conclusions This study identified the upstream signalling mediators that contribute to SHP-2 dependent monocyte activation in T2DM conditions. Glucose metabolite (MG) or RAGE ligand (AGE-BSA) alone were sufficient to induce a pro-migratory phenotype in monocytes by upregulating SHP-2. Of note, an inflammatory state seems to accelerate this effect since enhanced TNFα levels were found to be positively correlated with the augmented SHP-2 expression. Moreover, we identified the RAGE-NFκB signalling axis through which the SHP-2 upregulation is conveyed when augmented accumulation of glucose metabolites occur. These findings reveal a basis for potential new therapeutic approaches to prevent accelerated CAD and PAD in diabetic patients since independent pharmacological inhibition of every step in the RAGE-NFκB-SHP-2 axis was sufficient to reset the aberrant monocyte activation. FUNDunding Acknowledgement Type of funding sources: Other. Main funding source(s): Interdisciplinary Center for Clinical Research of the Medical Faculty of the University of Münster

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Extracellular Matrix Remodeling in the Retina and Optic Nerve of a Novel Glaucoma Mouse Model

Biology ◽

10.3390/biology10030169 ◽

2021 ◽

Vol 10 (3) ◽

pp. 169

Author(s):

Jacqueline Reinhard ◽

Susanne Wiemann ◽

Sebastian Hildebrandt ◽

Andreas Faissner

Keyword(s):

Extracellular Matrix ◽

Optic Nerve ◽

Protein Tyrosine Phosphatase ◽

Tyrosine Phosphatase ◽

Nerve Fibers ◽

Receptor Protein ◽

Mrna Levels ◽

Protein Tyrosine ◽

Tenascin C ◽

Iop Elevation

Glaucoma is a neurodegenerative disease that is characterized by the loss of retinal ganglion cells (RGC) and optic nerve fibers. Increased age and intraocular pressure (IOP) elevation are the main risk factors for developing glaucoma. Mice that are heterozygous (HET) for the mega-karyocyte protein tyrosine phosphatase 2 (PTP-Meg2) show chronic and progressive IOP elevation, severe RGCs loss, and optic nerve damage, and represent a valuable model for IOP-dependent primary open-angle glaucoma (POAG). Previously, evidence accumulated suggesting that glaucomatous neurodegeneration is associated with the extensive remodeling of extracellular matrix (ECM) molecules. Unfortunately, little is known about the exact ECM changes in the glaucomatous retina and optic nerve. Hence, the goal of the present study was to comparatively explore ECM alterations in glaucomatous PTP-Meg2 HET and control wild type (WT) mice. Due to their potential relevance in glaucomatous neurodegeneration, we specifically analyzed the expression pattern of the ECM glycoproteins fibronectin, laminin, tenascin-C, and tenascin-R as well as the proteoglycans aggrecan, brevican, and members of the receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) family. The analyses were carried out in the retina and optic nerve of glaucomatous PTP-Meg2 HET and WT mice using quantitative real-time PCR (RT-qPCR), immunohistochemistry, and Western blot. Interestingly, we observed increased fibronectin and laminin levels in the glaucomatous HET retina and optic nerve compared to the WT group. RT-qPCR analyses of the laminins α4, β2 and γ3 showed an altered isoform-specific regulation in the HET retina and optic nerve. In addition, an upregulation of tenascin-C and its interaction partner RPTPβ/ζ/phosphacan was found in glaucomatous tissue. However, comparable protein and mRNA levels for tenascin-R as well as aggrecan and brevican were observed in both groups. Overall, our study showed a remodeling of various ECM components in the glaucomatous retina and optic nerve of PTP-Meg2 HET mice. This dysregulation could be responsible for pathological processes such as neovascularization, inflammation, and reactive gliosis in glaucomatous neurodegeneration.

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Crocodile blood supplementation protects vascular function in diabetic mice

Food Production, Processing and Nutrition ◽

10.1186/s43014-021-00066-w ◽

2021 ◽

Vol 3 (1) ◽

Author(s):

Chui Yiu Bamboo Chook ◽

Francis M. Chen ◽

Gary Tse ◽

Fung Ping Leung ◽

Wing Tak Wong

Keyword(s):

Endothelial Cells ◽

Vascular Function ◽

Quantitative Polymerase Chain Reaction ◽

Functional Study ◽

Diabetic Patients ◽

Diabetic Mice ◽

Gene Expressions ◽

Inflammatory State ◽

Crocodile Blood

Abstract Cardiovascular disease is a major cause of mortality in diabetic patients due to the heightened oxidative stress and pro-inflammatory state in vascular tissues. Effective approaches targeting cardiovascular health for diabetic patients are urgently needed. Crocodile blood, an emerging dietary supplement, was suggested to have anti-oxidative and anti-inflammatory effects in vitro, which have yet to be proven in animal models. This study thereby aimed to evaluate whether crocodile blood can protect vascular function in diabetic mice against oxidation and inflammation. Diabetic db/db mice and their counterparts db/m+ mice were treated daily with crocodile blood soluble fraction (CBSF) or vehicle via oral gavage for 4 weeks before their aortae were harvested for endothelium-dependent relaxation (EDR) quantification using wire myograph, which is a well-established functional study for vascular function indication. Organ culture experiments culturing mouse aortae from C57BL/6 J mice with or without IL-1β and CBSF were done to evaluate the direct effect of CBSF on endothelial function. Reactive oxygen species (ROS) levels in mouse aortae were assessed by dihydroethidium (DHE) staining with inflammatory markers in endothelial cells quantified by quantitative polymerase chain reaction (qPCR). CBSF significantly improved deteriorated EDR in db/db diabetic mice through both diet supplementation and direct culture, with suppression of ROS level in mouse aortae. CBSF also maintained EDR and reduced ROS levels in mouse aortae against the presence of pro-inflammatory IL-1β. Under the pro-inflammatory state induced by IL-1β, gene expressions of inflammatory cytokines were downregulated, while the protective transcripts UCP2 and SIRT6 were upregulated in endothelial cells. Our study suggests a novel beneficial effect of crocodile blood on vascular function in diabetic mice and that supplementation of diet with crocodile blood may act as a complementary approach to protect against vascular diseases through anti-oxidation and anti-inflammation in diabetic patients. Graphical abstract

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Credentialing and Pharmacologically Targeting PTP4A3 Phosphatase as a Molecular Target for Ovarian Cancer

Biomolecules ◽

10.3390/biom11070969 ◽

2021 ◽

Vol 11 (7) ◽

pp. 969

Author(s):

John S. Lazo ◽

Elizabeth R. Sharlow ◽

Robert Cornelison ◽

Duncan J. Hart ◽

Danielle C. Llaneza ◽

...

Keyword(s):

Ovarian Cancer ◽

Cell Migration ◽

Tyrosine Phosphatase ◽

In Vitro Cytotoxicity ◽

Mrna Levels ◽

High Grade ◽

Drug Resistant ◽

Ovca Cell

High grade serous ovarian cancer (OvCa) frequently becomes drug resistant and often recurs. Consequently, new drug targets and therapies are needed. Bioinformatics-based studies uncovered a relationship between high Protein Tyrosine Phosphatase of Regenerating Liver-3 (PRL3 also known as PTP4A3) expression and poor patient survival in both early and late stage OvCa. PTP4A3 mRNA levels were 5–20 fold higher in drug resistant or high grade serous OvCa cell lines compared to nonmalignant cells. JMS-053 is a potent allosteric small molecule PTP4A3 inhibitor and to explore further the role of PTP4A3 in OvCa, we synthesized and interrogated a series of JMS-053-based analogs in OvCa cell line-based phenotypic assays. While the JMS-053 analogs inhibit in vitro PTP4A3 enzyme activity, none were superior to JMS-053 in reducing high grade serous OvCa cell survival. Because PTP4A3 controls cell migration, we interrogated the effect of JMS-053 on this cancer-relevant process. Both JMS-053 and CRISPR/Cas9 PTP4A3 depletion blocked cell migration. The inhibition caused by JMS-053 required the presence of PTP4A3. JMS-053 caused additive or synergistic in vitro cytotoxicity when combined with paclitaxel and reduced in vivo OvCa dissemination. These results indicate the importance of PTP4A3 in OvCa and support further investigations of the lead inhibitor, JMS-053.

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Increased C-reactive protein concentrations in patients admitted to the emergency department with troponin level elevations decreases the probability of myocardial ischemia

European Heart Journal Acute Cardiovascular Care ◽

10.1093/ehjacc/zuab020.057 ◽

2021 ◽

Vol 10 (Supplement_1) ◽

Author(s):

SR Meisel ◽

OM Kobo ◽

M Kleisener Shochat ◽

M Saada ◽

N Amsalem ◽

...

Keyword(s):

Emergency Department ◽

Myocardial Ischemia ◽

Cardiac Injury ◽

Medical Problem ◽

Troponin Level ◽

Elevated Troponin ◽

Reactive Protein ◽

Funding Sources ◽

Cardiology Department ◽

Inflammatory State

Abstract Funding Acknowledgements Type of funding sources: None. Background Patients frequently present to the emergency department (ED) with chest pain, dyspnea, or other symptoms with elevated troponin level. This finding prompts a provisional diagnosis of myocardial ischemia and raises the need to exclude this possibility. However, elevated blood troponin may be the result of a systemic inflammatory or infectious state merely representing cardiac injury and not myocardial ischemia. Purpose We hypothesized that the ratio of CRP/troponin could reflect the extent of the systemic inflammatory state that induces an attendant cardiac injury, which if sufficiently high could exclude myocardial ischemia. Methods Study population included 10774 patients admitted to the ED during the years 2016-2019 with cTn level higher > 14 ng/liter. CRP level was measured in all patients and CRP/troponin ratio was assessed against discharge diagnosis of myocardial ischemia, in order to evaluate its ability to exclude ischemic etiology of symptoms. The incidence of myocardial ischemia among study patients decreased with increasing CRP/troponin value. Results The prevalence of myocardial ischemia was 760/2694 patients (28.2%), 415/2694 (15.4%), 294/2695 (10.9%) and 130/2694 (4.8%) with 1st-4th CRP/troponin quartile, respectively (p < 0.0001). Logistic regression has shown that the probability of myocardial ischemia decreased by 53%, 68%, and 87% in the second to fourth CRP/troponin quartile compared with the first quartile, respectively (p < 0.0001). Conclusion The present study has shown that increased CRP level seems to modulate the specificity of simultaneous troponin as a marker of ischemia. As CRP level increases, so increases the likelihood that concomitant elevated troponin is due to myocardial injury and not due to myocardial ischemia. The clinical implication is that in the presence of a high CRP/troponin ratio, admission to the cardiology department and coronary investigation are unnecessary, whereas appropriate investigation of the actual medical problem is warranted.

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Upregulation of PKC genes and isozymes in cardiovascular tissues during early stages of experimental diabetes

Physiological Genomics ◽

10.1152/physiolgenomics.00125.2002 ◽

2003 ◽

Vol 12 (2) ◽

pp. 139-146 ◽

Cited By ~ 49

Author(s):

Mingzhang Guo ◽

Mack H. Wu ◽

Ferenc Korompai ◽

Sarah Y. Yuan

Keyword(s):

Protein Expression ◽

Cardiovascular System ◽

Time Course ◽

Experimental Diabetes ◽

Pathological Condition ◽

Expression Profiles ◽

Diabetic Patients ◽

Mrna Levels ◽

Early Stages ◽

Protein Expression Profiles

The protein kinase C (PKC) pathway has recently been recognized as an important mechanism in the development of diabetic complications including cardiomyopathy and angiopathy. Although an increase in PKC kinase activity has been detected in the cardiovascular system of diabetic patients and animals, it is unclear whether the same pathological condition alters PKC at the transcriptional and translational levels. In this study we assessed quantitatively the mRNA and protein expression profiles of PKC isozymes in the heart and vascular tissues from streptozotocin-induced diabetic pigs. Partial regions of the porcine PKCα, β1, and β2 mRNAs were sequenced, and real-time RT-PCR assays were developed for PKC mRNA quantification. The results showed a significant increase in the mRNA levels of PKCα, β1, and β2 in the heart at 4–8 wk of diabetes. In concomitance, the PKCβ1 and β2 genes, but not the PKCα gene, were upregulated in the diabetic aorta. Correspondingly, there was a significant increase in the protein expression of PKCα and β2 in the heart and PKCβ2 in the aorta with a time course correlated to that of mRNA expression. In summary, PKCβ2 was significantly upregulated in the heart and aorta at both the transcriptional and translational levels during early stages of experimental diabetes, suggesting that PKCβ2 may be a prominent target of diabetic injury in the cardiovascular system.

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TWIST1 and TWIST2 regulate glycogen storage and inflammatory genes in skeletal muscle

Journal of Endocrinology ◽

10.1530/joe-14-0474 ◽

2015 ◽

Vol 224 (3) ◽

pp. 303-313 ◽

Cited By ~ 7

Author(s):

Jonathan M Mudry ◽

Julie Massart ◽

Ferenc L M Szekeres ◽

Anna Krook

Keyword(s):

Skeletal Muscle ◽

Metabolic Diseases ◽

Glycogen Synthesis ◽

C2c12 Cells ◽

Acid Oxidation ◽

Diabetic Patients ◽

Mrna Levels ◽

Intact Mouse ◽

Mouse Muscle ◽

The Impact

TWIST proteins are important for development of embryonic skeletal muscle and play a role in the metabolism of tumor and white adipose tissue. The impact of TWIST on metabolism in skeletal muscle is incompletely studied. Our aim was to assess the impact of TWIST1 and TWIST2 overexpression on glucose and lipid metabolism. In intact mouse muscle, overexpression of Twist reduced total glycogen content without altering glucose uptake. Expression of TWIST1 or TWIST2 reducedPdk4mRNA, while increasing mRNA levels ofIl6,Tnfα, andIl1β. Phosphorylation of AKT was increased and protein abundance of acetyl CoA carboxylase (ACC) was decreased in skeletal muscle overexpressing TWIST1 or TWIST2. Glycogen synthesis and fatty acid oxidation remained stable in C2C12 cells overexpressing TWIST1 or TWIST2. Finally, skeletal muscle mRNA levels remain unaltered inob/obmice, type 2 diabetic patients, or in healthy subjects before and after 3 months of exercise training. Collectively, our results indicate that TWIST1 and TWIST2 are expressed in skeletal muscle. Overexpression of these proteins impacts proteins in metabolic pathways and mRNA level of cytokines. However, skeletal muscle levels of TWIST transcripts are unaltered in metabolic diseases.

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T-cell mediated autoimmunity to the insulinoma-associated protein 2 islet tyrosine phosphatase in type 1 diabetes mellitus

Acta Endocrinologica ◽

10.1530/eje.0.1410272 ◽

1999 ◽

pp. 272-278 ◽

Cited By ~ 12

Author(s):

F Dotta ◽

S Dionisi ◽

V Viglietta ◽

C Tiberti ◽

MC Matteoli ◽

...

Keyword(s):

Cell Proliferation ◽

Type 1 Diabetes ◽

T Cell ◽

T Lymphocyte ◽

Tyrosine Phosphatase ◽

T Cell Response ◽

T Cell Proliferation ◽

Diabetic Patients ◽

Hla Dr

The target molecules of the T-cell response in type 1 diabetes, despite their pathogenic importance, remain largely uncharacterized, especially in humans. Interestingly, molecules such as insulin and glutamic acid decarboxylase (GAD) have been shown to be a target not only of autoantibodies, but also of autoreactive T-lymphocytes both in man and in the non-obese diabetic (NOD) mouse. In the present study we aimed to determine the existence of a specific T-cell response towards the insulinoma-associated protein 2 (IA-2) islet tyrosine phosphatase, a recently identified autoantigen which is the target of autoantibodies strongly associated with diabetes development. Human recombinant IA-2 produced in Escherichia coli, was tested for its reactivity with peripheral blood lymphocytes obtained from 16 newly diagnosed type 1 diabetic patients and from 25 normal controls, 15 of whom were HLA-DR-matched. A T-cell proliferation assay was performed in triplicate employing freshly isolated cells in the absence or in the presence of the antigen to be tested (at two different concentrations: 2 microg/ml and 10 microg/ml). A specific T-cell proliferation (defined as a stimulation index (S.I.) >/=3) was observed against IA-2 used at a concentration of 10 microg/ml (but not of 2 microg/ml) in 8/16 diabetic patients, in 1/15 HLA-DR-matched control subjects (P<0.01 by Fisher exact test) and in 0/10 of the remaining normal individuals. A statistically significant difference (P<0.003 by Mann-Whitney U test) was also observed in S.I. values between patients (3.1+/-1.4) and HLA-DR-matched controls (1.7+/-0.54) employing IA-2 at a concentration of 10 microg/ml. However, when IA-2 was used at a concentration of 2 microg/ml, the difference in S. I. between patients (1.65+/-0.8) and controls (1.0+/-0.3) did not reach statistical significance. In conclusion, these data show the presence of a specific, dose-dependent T-lymphocyte response against the IA-2 islet tyrosine phosphatase at the onset of type 1 diabetes. Consequently, this molecule appears to be a target not only at the B-lymphocyte but also at the T-lymphocyte level, reinforcing the potential pathogenic role of this autoantigen in the islet destructive process.

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Risk of atrial fibrillation development of sodium-glucose co-transporter 2 inhibitor and thiazolidinedione treatment among patients with type 2 diabetes

European Heart Journal ◽

10.1093/eurheartj/ehab724.0460 ◽

2021 ◽

Vol 42 (Supplement_1) ◽

Author(s):

Y.R Kim ◽

S.E Lee ◽

K.A Kim

Keyword(s):

Atrial Fibrillation ◽

Type 2 Diabetes ◽

Incidence Rates ◽

Good Choice ◽

Diabetic Patients ◽

Funding Sources ◽

Korean National Health Insurance ◽

Outcome Event

Abstract Background Type 2 diabetes is an independent risk factor for the development of atrial fibrillation (AF). Recently, SGLT-2i has been shown to decrease the incidence of AF through several mechanisms including reduction of atrial dilatation via diuresis and lowering body weight. On the other hand, the use of TZD was found to protect diabetic patients from new-onset AF in observational studies. Thus, we aimed to compare the effect of SGLT-2i and TZD on the risk of AF development. Methods Using the Korean National Health Insurance Service database, we included patients with type 2 diabetes who were prescribed SGLT-2i or TZD at least once from January 2014 to December 2018. Patients were followed until the outcome event, death, or 31 December 2018. Sensitivity analysis was performed only including patients who prescribed study drugs ≥90 days. Results A total of 206,986 patients were included (88,227 patients in SGLT-2i group and 118,759 in TZD group). Baseline characteristics were mean age was 57 years and 57.4% were male; mean body mass index was 26.3kg/m2 and 68.3% had hypertension. During follow-up, the incidence rates of AF were 1.36% in SGLT-2i-treated patients and 0.87% TZD-treated patients, respectively (p=0.0002). The hazard ratio (HR) of AF was 0.846 (95% confidence interval [CI]: 0.0.775–0.923) in SGLT-2i-treated patients compared with TZD-treated patients. Conclusions In this study, the risk of AF development was significantly lower in patients treated with SGLT-2i versus TZD. SGLT2 would be a good choice for the patients with high risk of AF development among diabetes mellitus. FUNDunding Acknowledgement Type of funding sources: None.

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Distinct serotypes of streptococcal M proteins mediate fibrinogen-dependent platelet activation and pro-inflammatory effects

Infection and Immunity ◽

10.1128/iai.00462-21 ◽

2021 ◽

Author(s):

Frida Palm ◽

Sounak Chowdhury ◽

Sara Wettemark ◽

Johan Malmström ◽

Lotta Happonen ◽

...

Keyword(s):

Platelet Activation ◽

M Protein ◽

Multiparameter Flow Cytometry ◽

Invasive Infection ◽

Monocyte Activation ◽

Platelet Surface ◽

Sequence Variations ◽

M Proteins ◽

Inflammatory State ◽

Life Threatening

Sepsis is a life-threatening complication of infection that is characterised by a dysregulated inflammatory state and disturbed hemostasis. Platelets are the main regulators of hemostasis, and they also respond to inflammation. The human pathogen Streptococcus pyogenes can cause local infection that may progress to sepsis. There are more than 200 different serotypes of S. pyogenes defined according to sequence variations in the M protein. The M1 serotype is among ten serotypes that are predominant in invasive infection. M1 protein can be released from the surface and has previously been shown to generate platelet, neutrophil and monocyte activation. The platelet dependent pro-inflammatory effects of other serotypes of M protein associated with invasive infection (M3, M5, M28, M49 and M89) is now investigated using a combination of multiparameter flow cytometry, ELISA, aggregometry and quantitative mass spectrometry. We demonstrate that only M1-, M3- and M5 protein serotypes can bind fibrinogen in plasma and mediate fibrinogen and IgG dependent platelet activation and aggregation, release of granule proteins, upregulation of CD62P to the platelet surface, and complex formation with neutrophils and monocytes. Neutrophil and monocyte activation, determined as upregulation of surface CD11b, is also mediated by M1-, M3- and M5 protein serotypes, while M28-, M49- or M89 proteins failed to mediate activation of platelets or leukocytes. Collectively, our findings reveal novel aspects of the immunomodulatory role of fibrinogen acquisition and platelet activation during streptococcal infections.

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Abstract MP123: The Fate and Role of Pericytes in Repair and Remodeling of the Infarcted Heart

Circulation Research ◽

10.1161/res.127.suppl_1.mp123 ◽

2020 ◽

Vol 127 (Suppl_1) ◽

Author(s):

Linda Alex ◽

Ya Su ◽

Nikolaos G Frangogiannis

Keyword(s):

Lineage Tracing ◽

Mrna Levels ◽

Infarct Zone ◽

Subsequent Formation ◽

Mural Cells ◽

Infarcted Heart ◽

Reporter Mice ◽

Ng2 Cells ◽

Migratory Phenotype

Repair of the infarcted heart is dependent on inflammation-driven activation of myofibroblasts (MFs) and subsequent formation of a scar. Though pericytes have been implicated in injury-associated fibroblast activation in several organs, their potential role in cardiac repair and fibrosis has not been studied. We hypothesized that myocardial infarction (MI) may induce pericyte activation, contributing to repair through pericyte to MF conversion, secretion of fibrogenic mediators, or regulation of angiogenesis. In order to test the hypothesis, we generated pericyte/fibroblast reporter mice (NG2 DsRed ;PDGFRα GFP ). In normal myocardium, NG2 labeled peri-endothelial mural cells that coexpressed PDGFRβ, whereas PDGFRα identified interstitial cells with fibroblast characteristics. Pericytes and fibroblasts had distinct transcriptomic profiles: NG2+/PDGFRα- pericytes expressed αSMA and low amounts of extracellular matrix (ECM) genes, whereas PDGFRα+/NG2- fibroblasts synthesized collagens. Pericyte rarefaction was noted in the necrotic core 3 days after non-reperfused MI. 3-7 days post MI, expansion of the NG2+ population in the infarct zone was associated with emergence of non-mural NG2+/αSMA+ cells with MF characteristics. FACS-sorted NG2+/PDGFRα- cells from 7-day infarcts expressed higher levels of ColIα2 (7.2±1.0-fold) and ColIIIα1 (8.9±1.14-fold), when compared to NG2+/PDGFRα- cells from normal hearts. NG2+ cells had high mRNA levels of integrins α1, αV, β1, and β5, and of MMP14, reflecting an activated migratory phenotype. To examine whether expression of ECM genes by infarct pericytes is due to fibroblast conversion, we did lineage tracing studies using NG2CreER TM ;Rosa tdTomato mice bred with the PDGFRα GFP line for reliable fibroblast identification. 7 days post MI, 5.7%±1.04 of PDGFRα+ fibroblasts were derived from NG2+ cells. Also, αSMA staining showed that 10.49%±2.73 of infarct MFs were derived from NG2+ lineage. The majority of mural cells wrapping neovessels were derived from NG2+ cells, suggesting a role for resident pericytes in infarct angiogenesis. In conclusion, upon MI, pericytes become activated and contribute to repair by undergoing conversion to a subset of myofibroblasts and by coating infarct neovessels.

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