Differential expression of immunomodulatory galectin-1 in peripheral leukocytes and adult tissues and its cytosolic organization in striated muscle

Galectin-1 (Gal-1) is important in immune function and muscle regeneration, but its expression and localization in adult tissues and primary leukocytes remain unclear. To address this, we generated a specific monoclonal antibody against Gal-1, termed alpha hGal-1, and defined a sequential peptide epitope that it recognizes, which is preserved in human and porcine Gal-1, but not in murine Gal-1. Using alpha hGal-1, we found that Gal-1 is expressed in a wide range of porcine tissues, including striated muscle, liver, lung, brain, kidney, spleen, and intestine. In most types of cells, Gal-1 exhibits diffuse cytosolic expression, but in cells within the splenic red pulp, Gal-1 showed both cytosolic and nuclear localization. Gal-1 was also expressed in arterial walls and exhibited prominent cytosolic and nuclear staining in cultured human endothelial cells. However, human peripheral leukocytes and promyelocytic HL60 cells lack detectable Gal-1 and also showed very low levels of Gal-1 mRNA. In striking contrast, Gal-1 exhibited an organized cytosolic staining pattern within striated muscle tissue of cardiac and skeletal muscle and colocalized with sarcomeric actin on I bands. These results provide insights into previously defined roles for Gal-1 in inflammation, immune regulation and muscle biology.

Gender-specific vascular effects elicited by chronic ethanol consumption in rats: a role for inducible nitric oxide synthase

Background and purpose: Epidemiological data suggest that the risk of ethanol-associated cardiovascular disease is greater in men than in women. This study investigates the mechanisms underlying gender-specific vascular effects elicited by chronic ethanol consumption in rats. Experimental approach: Vascular reactivity experiments using standard muscle bath procedures were performed on isolated thoracic aortae from rats. mRNA and protein for inducible NO synthase (iNOS) and for endothelial NOS (eNOS) was assessed by RT-PCR or western blotting, respectively. Key results: In male rats, chronic ethanol consumption enhanced phenylephrine-induced contraction in both endothelium-intact and denuded aortic rings. However, in female rats, chronic ethanol consumption enhanced phenylephrine-induced contraction only in endothelium denuded aortic rings. After pre-incubation of endothelium-intact rings with L-NAME, both male and female ethanol-treated rats showed larger phenylephrine-induced contractions in aortic rings, compared to the control group. Acetylcholine-induced relaxation was not affected by ethanol consumption. The effects of ethanol on responses to phenylephrine were similar in ovariectomized (OVX) and intact (non-OVX) female rats. In the presence of aminoguanidine, but not 7-nitroindazole, the contractions to phenylephrine in rings from ethanol-treated female rats were greater than that found in control tissues in the presence of the inhibitors. mRNA levels for eNOS and iNOS were not altered by ethanol consumption. Ethanol intake reduced eNOS protein levels and increased iNOS protein levels in aorta from female rats. Conclusions and implications: Gender differences in the vascular effects elicited by chronic ethanol consumption were not related to ovarian hormones but seemed to involve the upregulation of iNOS.

Expression analysis of putative vitellogenin and lipophorin receptors in honey bee (Apis mellifera L.) queens and workers

Two members of the low density lipoprotein receptor (LDLR) family were identified as putative orthologs for a vitellogenin receptor (Amvgr) and a lipophorin receptor (Amlpr) in the Apis mellifera genome. Both receptor sequences have the structural motifs characteristic of LDLR family members and show a high degree of similarity with sequences of other insects. RT-PCR analysis of Amvgr and Amlpr expression detected the presence of both transcripts in different tissues of adult female (ovary, fat body, midgut, head and specifically hypopharyngeal gland), as well as in embryos. In the head RNA samples we found two variant forms of AmLpR: a full length one and a shorter one lacking 29 amino acids in the O-linked sugar domain. In ovaries the expression levels of the two honey bee LDLR members showed opposing trends: whereas Amvgr expression was upregulated as the ovaries became activated, Amlpr transcript levels gradually declined. In situ hybridization analysis performed on ovaries detected Amvgr mRNA exclusively in germ line cells and corroborated the qPCR results showing an increase in Amvgr gene expression concomitant with follicle growth. (C) 2008 Elsevier Ltd. All rights reserved.

TRADE-OFF BETWEEN IMMUNE STIMULATION AND EXPRESSION OF STORAGE PROTEIN GENES

Proteins stored in insect hemolymph may serve (is a source of amino acids and energy for metabolism, and development. The expression of the main storage proteins was assessed in bacterial-challenged honey bees using real-time (RT)-PCH and Western blot.. After ensuring that. the immune system had, been activated by measuring the ensuing expression (, the innate immune response genes, defensin-1 (def-1) and prophenoloxidase (pro PO), we verified the expression of four genes encoding storage proteins. The levels of vitellogenin (vg) mRNA and of the respective protein. were significantly lowered in bees injected with bacteria or water only (injury). An equivalent response was observed in orally-infected bees. The levels of apolipophorin II/I (apoLP-II/I) and hexamerin (hex 70a) mRNAs did not significantly change, but levels of Hex 70a protein subunit showed a substantial decay after bacterial challenge or injury. Infection also caused a strong reduction in the levels of apoLP-III transcripts. Our findings are consistent with a down-regulation, of the express and accumulation of storage proteins as a consequence of activation of the immune system, suggesting that this phenomenon. represents a strategy to redirect resources to combat injury or infection. (C) 2009 Wiley Periodicals, Inc.

Detrusor overactivity is associated with downregulation of large-conductance calcium- and voltage-activated potassium channel protein

Chang S, Gomes CM, Hypolite JA, Marx J, Alanzi J, Zderic SA, Malkowicz B, Wein AJ, Chacko S. Detrusor overactivity is associated with downregulation of large-conductance calcium-and voltage-activated potassium channel protein. Am J Physiol Renal Physiol 298: F1416-F1423, 2010. First published April 14, 2010; doi: 10.1152/ajprenal.00595.2009.-Large-conductance voltage-and calcium-activated potassium (BK) channels have been shown to play a role in detrusor overactivity (DO). The goal of this study was to determine whether bladder outlet obstructioninduced DO is associated with downregulation of BK channels and whether BK channels affect myosin light chain 20 (MLC(20)) phosphorylation in detrusor smooth muscle (DSM). Partial bladder outlet obstruction (PBOO) was surgically induced in male New Zealand White rabbits. The rabbit PBOO model shows decreased voided volumes and increased voiding frequency. DSM from PBOO rabbits also show enhanced spontaneous contractions compared with control. Both BK channel alpha- and beta-subunits were significantly decreased in DSM from PBOO rabbits. Immunostaining shows BK beta mainly expressed in DSM, and its expression is much less in PBOO DSM compared with control DSM. Furthermore, a translational study was performed to see whether the finding discovered in the animal model can be translated to human patients. The urodynamic study demonstrates several overactive DSM contractions during the urine-filling stage in benign prostatic hyperplasia (BPH) patients with DO, while DSM is very quiet in BPH patients without DO. DSM biopsies revealed significantly less BK channel expression at both mRNA and protein levels. The degree of downregulation of the BK beta-subunit was greater than that of the BK alpha-subunit, and the downregulation of BK was only associated with DO, not BPH. Finally, the small interference (si) RNA-mediated downregulation of the BK beta-subunit was employed to study the effect of BK depletion on MLC(20) phosphorylation. siRNA-mediated BK channel reduction was associated with an increased MLC(20) phosphorylation level in cultured DSM cells. In summary, PBOO-induced DO is associated with downregulation of BK channel expression in the rabbit model, and this finding can be translated to human BPH patients with DO. Furthermore, downregulation of the BK channel may contribute to DO by increasing the basal level of MLC(20) phosphorylation.

Prolonged recruitment manoeuvre improves lung function with less ultrastructural damage in experimental mild acute lung injury

The effects of prolonged recruitment manoeuvre (PRM) were compared with sustained inflation (SI) in paraquat-induced mild acute lung injury (ALI) in rats. Twenty-four hours after ALI induction, rats were anesthetized and mechanically ventilated with VT = 6 ml/kg and positive end-expiratory pressure (PEEP) = 5 cmH(2)O for 1 h. SI was performed with an instantaneous pressure increase of 40 cmH(2)O that was sustained for 40 s, while PRM was done by a step-wise increase in positive inspiratory pressure (PIP) of 15-20-25 cmH(2)O above a PEEP of 15 cm H(2)O (maximal PIP = 40 cmH(2)O), with interposed periods of PIP = 10 cmH(2)O above a PEEP = 15 cmH(2)O. Lung static elastance and the amount of alveolar collapse were more reduced with PRM than SI, yielding improved oxygenation. Additionally, tumour necrosis factor-alpha, interleukin-6, interferon-gamma, and type III procollagen mRNA expressions in lung tissue and lung epithelial cell apoptosis decreased more in PRM. In conclusion, PRM improved lung function, with less damage to alveolar epithelium, resulting in reduced pulmonary injury. (C) 2009 Elsevier BLV. All rights reserved.

Accentuated osteoclastic response to parathyroid hormone undermines bone mass acquisition in osteonectin-null mice

Matricellular proteins play a unique role in the skeleton as regulators of bone remodeling, and the matricellular protein osteonectin (SPARC, BM-40) is the most abundant non-collagenous protein in bone In. the absence of osteonectin, mice develop progressive low turnover osteopenia, particularly affecting trabecular bone. Polymorphisms in a regulatory region of the osteonectin gene are associated with bone mass in a subset of idiopathic osteoporosis patients, and these polymorphisms likely regulate osteonectin expression. Thus it is important to determine how osteonectin gene dosage affects skeletal function. Moreover, intermittent administration of parathyroid hormone (PTH) (1-34) is the only anabolic therapy approved for the treatment of osteoporosis, and it is critical to understand how modulators of bone remodeling, such as osteonectin, affect skeletal response to anabolic agents. In this study, 10 week old female wild type, osteonectin-haploinsufficient, and osteonectin-null mice (C57Bl/6 genetic background) were given 80 mu g/kg body weight/day PTH(1-34) for 4 weeks. Osteonectin gene dosage had a profound effect on bone microarchitecture. The connectivity density of trabecular bone in osteonectin-haploinsufficient mice was substantially decreased compared with that of wild type mice, suggesting compromised mechanical properties. Whereas mice of each genotype had a similar osteoblastic response to PTH treatment, the osteoclastic response was accentuated in osteonectin-haploinsufficient and osteonectin-null mice. Eroded surface and osteoclast number were significantly higher in PTH-treated osteonectin-null mice, as was endosteal area. In vitro studies confirmed that PTH induced the formation of more osteoclast-like cells in marrow from osteonectin-null mice compared with wild type. PTH treated osteonectin-null bone marrow cells expressed more RANKL mRNA compared with wild type. However, the ratio of RANKL:OPG mRNA was somewhat lower in PTH treated osteonectin-null cultures. Increased expression of RANKL in response to PTH could contribute to the accentuated osteoclastic response in osteonectin(-/-) mice, but other mechanisms are also likely to be involved. The molecular mechanisms by which PTH elicits bone anabolic vs. bone catabolic effects remain poorly understood. Our results imply that osteonectin levels may play a role in modulating the balance of bone formation and resorption in response to PTH. (c) 2008 Elsevier Inc. All rights reserved.

The therapeutic potential of recombinant BCG expressing the antigen S1PT in the intravesical treatment of bladder cancer

Purpose: Bacillus Calmette-Guerin (BCG) continues to be employed as the most effective immunotherapy against superficial bladder cancer. We have developed an rBCG-S1PT strain that induces a stronger cellular immune response than BCG. This preclinical study was designed to test the potential of rBCG-S1PT as an immunotherapeutic agent for intravesical bladder cancer therapy. Materials and methods: A tumor was induced in C57BL/6 mice after chemical cauterization of the bladder and inoculation of the tumor cell line MB49. Next, mice were treated by intravesical instillation with BCG, rBCG-S1PT, or PBS once a week for 4 weeks. After 35 days, the bladders were removed and weighed, Th1 (IL-2, IL-12, INOS, INF-gamma, TNF-alpha), and Th2 (IL-5, IL-6, IL-10, TGF-beta) cytokine mRNA responses in individual mice bladders were measured by quantitative real time PCR, and the viability of MB49 cells in 18-hour coculture with splenocytes from treated mice was assessed. In an equivalent experiment, animals were observed for 60 days to quantify their survival. Results: Both BCG and rBCG-S1PT immunotherapy resulted in bladder weight reduction, and rBCG-S1PT increased survival time compared with the control group. There were increases in TNF-alpha in the BCG treated group, as well as increases in TNF-alpha and IL-10 mRNA in the rBCG-S1PT group. The viability of MB49 cells cocultured with splenocytes from rBCG-S1PT-treated mice was lower than in both the BCG and control groups. Conclusions: rBCG-S1PT therapy improved outcomes and lengthened survival times. These results indicate that rBCG could serve as a useful substitute for wild-type BCG. (C) 2010 Elsevier Inc. All rights reserved.

Contractile activity per se induces transcriptional activation of SLC2A4 gene in soleus muscle: involvement of MEF2D, HIF-1a, and TR alpha transcriptional factors

Lima GA, Anhe GF, Giannocco G, Nunes MT, Correa-Giannella ML, Machado UF. Contractile activity per se induces transcriptional activation of SLC2A4 gene in soleus muscle: involvement of MEF2D, HIF-1a, and TR alpha transcriptional factors. Am J Physiol Endocrinol Metab 296: E132-E138, 2009. First published October 28, 2008; doi: 10.1152/ajpendo.90548.2008.-Skeletal muscle is a target tissue for approaches that can improve insulin sensitivity in insulin-resistant states. In muscles, glucose uptake is performed by the GLUT-4 protein, which is encoded by the SLC2A4 gene. SLC2A4 gene expression increases in response to conditions that improve insulin sensitivity, including chronic exercise. However, since chronic exercise improves insulin sensitivity, the increased SLC2A4 gene expression could not be clearly attributed to the muscle contractile activity per se and/or to the improved insulin sensitivity. The present study was designed to investigate the role of contractile activity per se in the regulation of SLC2A4 gene expression as well as in the participation of the transcriptional factors myocyte enhancer factor 2D (MEF2D), hypoxia inducible factor 1a (HIF-1a), and thyroid hormone receptor-alpha (TR alpha). The performed in vitro protocol excluded the interference of metabolic, hormonal, and neural effects. The results showed that, in response to 10 min of electrically induced contraction of soleus muscle, an early 40% increase in GLUT-4 mRNA (30 min) occurred, with a subsequent 65% increase (120 min) in GLUT-4 protein content. EMSA and supershift assays revealed that the stimulus rapidly increased the binding activity of MEF2D, HIF-1a, and TR alpha into the SLC2A4 gene promoter. Furthermore, chromatin immunoprecipitation assay confirmed, in native nucleosome, that contraction induced an approximate fourfold (P < 0.01) increase in MEF2D and HIF-1a-binding activity. In conclusion, muscle contraction per se enhances SLC2A4 gene expression and that involves MEF2D, HIF-1a, and TR alpha transcription factor activation. This finding reinforces the importance of physical activity to improve glycemic homeostasis independently of other additional insulin sensitizer approaches.

Aerobic Exercise Improves Reverse Cholesterol Transport in Cholesteryl Ester Transfer Protein Transgenic Mice

We analyzed the effect of a 6-week aerobic exercise training program on the in vivo macrophage reverse cholesterol transport (RCT) in human cholesteryl ester transfer protein (CETP) transgenic (CETP-tg) mice. Male CETP-tg mice were randomly assigned to a sedentary group or a carefully supervised exercise training group (treadmill 15 m/min, 30 min sessions, five sessions per week). The levels of plasma lipids were determined by enzymatic methods, and the lipoprotein profile was determined by fast protein liquid chromatography (FPLC). CETP activity was determined by measuring the transfer rate of (14)C-cholesterol from HDL to apo-B containing lipoproteins, using plasma from CETP-tg mice as a source of CETP. The reverse cholesterol transport was determined in vivo by measuring the [(3)H]-cholesterol recovery in plasma and feces (24 and 48 h) and in the liver (48 h) following a peritoneal injection of [(3)H]-cholesterol labeled J774-macrophages into both sedentary and exercise trained mice. The protein levels of liver receptors were determined by immunoblot, and the mRNA levels for liver enzymes were measured using RT-PCR. Exercise training did not significantly affect the levels of plasma lipids or CETP activity. The HDL fraction assessed by FPLC was higher in exercise-trained compared to sedentary mice. In comparison to the sedentary group, a greater recovery of [(3)H]-cholesterol from the injected macrophages was found in the plasma, liver and feces of exercise-trained animals. The latter occurred even with a reduction in the liver CYP7A1 mRNA level in exercised trained animals. Exercise training increased the liver LDL receptor and ABCA-1 protein levels, although the SR-BI protein content was unchanged. The RCT benefit in CETP-tg mice elicited by exercise training helps to elucidate the role of exercise in the prevention of atherosclerosis in humans.

PPAR gamma expression is increased in systemic lupus erythematosus patients and represses CD40/CD40L signaling pathway

Systemic lupus erythematosus (SLE) is a heterogeneous disease involving several immune cell types and pro-inflammatory signals, including the one triggered by binding of CD40L to the receptor CD40. Peroxisome-proliferator activated receptor gamma (PPAR gamma) is a transcription factor with anti-inflammatory properties. Here we investigated whether CD40 and PPAR gamma could exert opposite effects in the immune response and the possible implications for SLE. Increased PPAR gamma mRNA levels were detected by real-time PCR in patients with active SLE, compared to patients with inactive SLE PPAR gamma/GAPDH mRNA = 2.21 +/- 0.49 vs. 0.57 +/- 0.14, respectively (p < 0.05) or patients with infectious diseases and healthy subjects (p < 0.05). This finding was independent of the corticosteroid therapy. We further explored these observations in human THP1 and in SLE patient-derived macrophages, where activation of CD40 by CD40L promoted augmented PPAR gamma gene transcription compared to non-stimulated cells (PPAR gamma/GAPDH mRNA = 1.14 +/- 0.38 vs. 0.14 +/- 0.01, respectively; p < 0.05). This phenomenon occurred specifically upon CD40 activation, since lipopolysaccharide treatment did not induce a similar response. In addition, increased activity of PPAR gamma was also detected after CD40 activation, since higher PPAR gamma-dependent transcription of CD36 transcription was observed. Furthermore, CD40L-stimulated transcription of CD80 gene was elevated in cells treated with PPAR gamma-specific small interfering RNA (small interfering RNA, siRNA) compared to cells treated with CD40L alone (CD80/GAPDH mRNA = 0.11 +/- 0.04 vs. 0.05 +/- 0.02, respectively; p < 0.05), suggesting a regulatory role for PPAR gamma on the CD40/CD40L pathway. Altogether, our findings outline a novel mechanism through which PPAR gamma regulates the inflammatory signal initiated by activation of CD40, with important implications for the understanding of immunological mechanisms underlying SLE and the development of new treatment strategies. Lupus (2011) 20, 575-587.

Gene expression reprogramming protects macrophage from septic-induced cell death

Sepsis induces a systemic inflammatory response leading to tissue damage and cell death. LPS tolerance affects inflammatory response. To comprehend potential new mechanisms of immune regulation in endotoxemia, we examined macrophage mRNA expression by macroarray affected by LPS tolerance. LPS tolerance was induced with subcutaneous administration of 1 mg/kg/day of LPS over 5 days. Macrophages were isolated from the spleen and the expression of 1200 genes was quantitatively analyzed by the macroarray technique. The tolerant group displayed relevant changes in the expression of 84 mRNA when compared to naive mice. A functional group of genes related to cell death regulation was identified. PARP-1, caspase 3, FASL and TRAIL genes were confirmed by RT-PCR to present lower expression in tolerant mice. In addition, reduced expression of the pro-inflammatory genes TNF-alpha and IFN-gamma in the tolerant group was demonstrated. Following this, animals were challenged with polymicrobial sepsis. Flow cytometry analysis showed reduced necrosis and apoptosis in macrophages from the tolerant group compared to the naive group. Finally, a survival study showed a significant reduction in mortality in the tolerant group. Thus, in the current study we provide evidence for the selective reprogramming of the gene expression of cell death pathways during LPS tolerance and link these changes to protection from cell death and enhanced survival rates. (C) 2010 Elsevier Ltd. All rights reserved.

Endotoxin tolerance: Selective alterations in gene expression and protection against lymphocyte death

Extensive lymphocyte apoptosis may be an important cause of immune suppression in sepsis. Here we investigated the effect of LPS tolerance on lymphocyte apoptosis in an experimental model of polymicrobial infection. Tolerance was induced by the injection of lipopolysaccharide (1.0 mg/kg/subcutaneously) once a day for 5 days. Macroarray analysis of mRNA isolated from T-(CD4) lymphocytes was used to identify genes that are differentially expressed during LPS tolerance. In addition, assessment of the expression of apoptosis-associated lymphocyte gene products and apoptotic events was performed on the 8th day; 6 h after the terminal challenge with polymicrobial infection or high-dose LPS administration. Survival studies with polymicrobial infection were also conducted. LPS tolerance induced a broad reprogramming of cell death pathways, including a suppression of receptor-mediated and mitochondrial apoptotic pathways, inflammatory caspases, alternate apoptotic pathways, as well as reduced expression of genes involved in necrosis. These alterations led to a marked resistance of lymphocytes against cell death during the subsequent period of sepsis. In addition, LPS tolerance produced an increased differentiation of T-lymphocytes to T(H)1 and T(H)2, with a T(H)1 differentiation predominance. Thus, in the current study we provide an evidence for a marked reprogramming of gene expression of multiple cell death pathways during LPS tolerance. These alterations may play a significant role in the observed protection of the animals from a subsequent lethal polymicrobial sepsis challenge. (C) 2009 Elsevier GmbH. All rights reserved.

Intake of trans Fatty Acids Causes Nonalcoholic Steatohepatitis and Reduces Adipose Tissue Fat Content

We investigated the effects of dietary trans fatty acids, PUFA, and SEA on body and liver fat content, liver histology, and mRNA of enzymes involved in fatty acid metabolism. LDL receptor knockout weaning male mice were fed for 16 wk with diets containing 40% energy as either trans fatty acids (TRANS), PUFA, or SEA. Afterwards, subcutaneous and epididymal fat were weighed and histological markers of nonalcoholic fatty liver disease (NAFLD) were assessed according to the Histological Scoring System for NAFLD. PPAR alpha, PPAR gamma, microsomal triglyceride transfer protein (MTP), carnitine palmitoyl transferase 1 (CPT-1), and sterol regulatory element binding protein-1c (SREBP-1c) mRNA were measured by quantitative RT-PCR. Food intake was similar in the 3 groups, although mice fed the TRANS diet gained less weight than those receiving the PUFA diet. Compared with the PUFA- and SEA-fed mice, TRANS-fed mice had greater plasma total cholesterol (TC) and triglyceride (TG) concentrations, less epididymal and subcutaneous fat, larger livers with nonalcoholic steatohepatitis (NASH)-like lesions, and greater liver TC and TG concentrations. Macrosteatosis in TRANS-fed mice was associated with a higher homeostasis model assessment of insulin resistance (HOMA(IR)) index and upregulated mRNA related to hepatic fatty acid synthesis (SREBP-1 c and PPAR gamma) and to downregulated MTP mRNA. Diet consumption did not alter hepatic mRNA related to fatty acid oxidation (PPAR alpha and CPT-1). In conclusion, compared with PUFA- and SFA-fed mice, TRANS-fed mice had less adiposity, impaired glucose tolerance characterized by greater HOMA(IR) index, and NASH-like lesions due to greater hepatic lipogenesis. These results demonstrate the role of trans fatty acid intake on the development of key features of metabolic syndrome. J. Nutr. 140: 1127-1132, 2010.

Aerobic Exercise Training-Induced Left Ventricular Hypertrophy Involves Regulatory MicroRNAs, Decreased Angiotensin-Converting Enzyme-Angiotensin II, and Synergistic Regulation of Angiotensin-Converting Enzyme 2-Angiotensin (1-7)

Aerobic exercise training leads to a physiological, nonpathological left ventricular hypertrophy; however, the underlying biochemical and molecular mechanisms of physiological left ventricular hypertrophy are unknown. The role of microRNAs regulating the classic and the novel cardiac renin-angiotensin (Ang) system was studied in trained rats assigned to 3 groups: (1) sedentary; (2) swimming trained with protocol 1 (T1, moderate-volume training); and (3) protocol 2 (T2, high-volume training). Cardiac Ang I levels, Ang-converting enzyme (ACE) activity, and protein expression, as well as Ang II levels, were lower in T1 and T2; however, Ang II type 1 receptor mRNA levels (69% in T1 and 99% in T2) and protein expression (240% in T1 and 300% in T2) increased after training. Ang II type 2 receptor mRNA levels (220%) and protein expression (332%) were shown to be increased in T2. In addition, T1 and T2 were shown to increase ACE2 activity and protein expression and Ang (1-7) levels in the heart. Exercise increased microRNA-27a and 27b, targeting ACE and decreasing microRNA-143 targeting ACE2 in the heart. Left ventricular hypertrophy induced by aerobic training involves microRNA regulation and an increase in cardiac Ang II type 1 receptor without the participation of Ang II. Parallel to this, an increase in ACE2, Ang (1-7), and Ang II type 2 receptor in the heart by exercise suggests that this nonclassic cardiac renin-angiotensin system counteracts the classic cardiac renin-angiotensin system. These findings are consistent with a model in which exercise may induce left ventricular hypertrophy, at least in part, altering the expression of specific microRNAs targeting renin-angiotensin system genes. Together these effects might provide the additional aerobic capacity required by the exercised heart. (Hypertension. 2011;58:182-189.).