Chronic diseases arise when there is mutual reinforcement of pathophysiological processes that cause an aberrant steady state. Such a sequence of events may underlie chronic constipation, which has been associated with dysbiosis of the gut. In this study we hypothesized that assemblage of microbial communities, directed by slow gastrointestinal transit, affects host function in a way that reinforces constipation and further maintains selection on microbial communities. In our study, we used two models – an opioid‐induced constipation model in mice, and a humanized mouse model where germ‐free mice were colonized with stool from a patient with constipation‐predominant irritable bowel syndrome (IBS‐C) in humans. We examined the impact of pharmacologically (loperamide)‐induced constipation (PIC) and IBS‐C on the structural and functional profile of the gut microbiota. Germ‐free (GF) mice were colonized with microbiota from PIC donor mice and IBS‐C patients to determine how the microbiota affects the host. PIC and IBS‐C promoted changes in the gut microbiota, characterized by increased relative abundance of Bacteroides ovatus and Parabacteroides distasonis in both models. PIC mice exhibited decreased luminal concentrations of butyrate in the cecum and altered metabolic profiles of the gut microbiota. Colonization of GF mice with PIC‐associated mice cecal or human IBS‐C fecal microbiota significantly increased GI transit time when compared to control microbiota recipients. IBS‐C‐associated gut microbiota also impacted colonic contractile properties. Our findings support the concept that constipation is characterized by disease‐associated steady states caused by reinforcement of pathophysiological factors in host‐microbe interactions.

Objective Nucleotide P2Y2 receptor (P2Y2R) contributes to vascular inflammation by increasing vascular cell adhesion molecule (VCAM)-1 expression in endothelial cells (EC), and global P2Y2R deficiency prevents fatty streak formation in ApoE−/− mice. Since P2Y2R is ubiquitously expressed in vascular cells, we investigated the contribution of endothelial P2Y2R in the pathogenesis of atherosclerosis. Approach and Results EC-specific P2Y2R-deficient mice were generated by breeding VEcadherin5-Cre mice with the P2Y2R “floxed” mice. Endothelial P2Y2R deficiency reduced eNOS activity and significantly altered ATP-and UTP-induced vasorelaxation without affecting vasodilatory responses to acetylcholine. Telemetric blood pressure and echocardiography measurements indicated that EC-specific P2Y2R-deficient mice did not develop hypertension. We investigated the role of endothelial P2Y2R in the development of atherosclerotic lesions by crossing the EC-specific P2Y2R knockout mice onto an ApoE−/− background and evaluated lesion development after feeding a standard chow diet for 25 weeks. Histopathological examination demonstrated reduced atherosclerotic lesions in the aortic sinus and entire aorta, decreased macrophage infiltration and increased smooth muscle cell and collagen content leading to the formation of a subendothelial fibrous cap in EC-specific P2Y2R-deficient ApoE−/− mice. Expression and proteolytic activity of matrix metalloproteinase (MMP)-2 was significantly reduced in atherosclerotic lesions from EC-specific P2Y2R-deficient ApoE−/− mice. Furthermore, EC-specific P2Y2R deficiency inhibited NO production leading to significant increase in SMC migration out of aortic explants. Conclusions EC-specific P2Y2R-deficiency reduces atherosclerotic burden and promotes plaque stability in ApoE−/− mice through impaired macrophage infiltration acting together with reduced MMP-2 activity and increased SMC migration.

Background and aims Mutations in the 5′-nucleotidase ecto (NT5E) gene that encodes CD73, a nucleotidase that converts AMP to adenosine, are linked to arterial calcification. However, the role of purinergic receptor signaling in the pathology of intimal calcification is not well understood. In this study, we examined whether extracellular nucleotides acting via P2Y2 receptor (P2Y2R) modulate arterial intimal calcification, a condition highly correlated with cardiovascular morbidity. Methods Apolipoprotein E, P2Y2R double knockout mice (ApoE−/−P2Y2R−/−) were used to determine the effect of P2Y2R deficiency on vascular calcification in vivo. Vascular smooth muscle cells (VSMC) isolated from P2Y2R−/− mice grown in high phosphate medium were used to assess the role of P2Y2R in the conversion of VSMC into osteoblasts. Luciferase-reporter assays were used to assess the effect of P2Y2R on the transcriptional activity of Runx2. Results P2Y2R deficiency in ApoE−/− mice caused extensive intimal calcification despite a significant reduction in atherosclerosis and macrophage plaque content. The ectoenzyme apyrase that degrades nucleoside di- and triphosphates accelerated high phosphate-induced calcium deposition in cultured VSMC. Expression of P2Y2R inhibits calcification in vitro inhibited the osteoblastic trans-differentiation of VSMC. Mechanistically, expression of P2Y2R inhibited Runx2 transcriptional activation of an osteocalcin promoter driven luciferase reporter gene. Conclusions This study reveals a role for vascular P2Y2R as an inhibitor of arterial intimal calcification and provides a new mechanistic insight into the regulation of the osteoblastic trans-differentiation of SMC through P2Y2R-mediated Runx2 antagonism. Given that calcification of atherosclerotic lesions is a significant clinical problem, activating P2Y2R may be an effective therapeutic approach for treatment or prevention of vascular calcification.

Objective- Unreliable antibodies often hinder the accurate detection of an endogenous protein, and this is particularly true for the cardiac and smooth muscle cofactor, MYOCD (myocardin). Accordingly, the mouse Myocd locus was targeted with 2 independent epitope tags for the unambiguous expression, localization, and activity of MYOCD protein. Approach and Results- 3cCRISPR (3-component clustered regularly interspaced short palindromic repeat) was used to engineer a carboxyl-terminal 3×FLAG or 3×HA epitope tag in mouse embryos. Western blotting with antibodies to each tag revealed a MYOCD protein product of ≈150 kDa, a size considerably larger than that reported in virtually all publications. MYOCD protein was most abundant in some adult smooth muscle-containing tissues with surprisingly low-level expression in the heart. Both alleles of Myocd are active in aorta because a 2-fold increase in protein was seen in mice homozygous versus heterozygous for FLAG-tagged Myocd. ChIP (chromatin immunoprecipitation)-quantitative polymerase chain reaction studies provide proof-of-principle data demonstrating the utility of this mouse line in conducting genome-wide ChIP-seq studies to ascertain the full complement of MYOCD-dependent target genes in vivo. Although FLAG-tagged MYOCD protein was undetectable in sections of adult mouse tissues, low-passaged vascular smooth muscle cells exhibited expected nuclear localization. Conclusions- This report validates new mouse models for analyzing MYOCD protein expression, localization, and binding activity in vivo and highlights the need for rigorous authentication of antibodies in biomedical research.

Background Lymphotoxin alpha (LTα) is expressed in human atherosclerotic lesions and genetic variations in the LTα pathway have been linked to myocardial infarction. Activation of the P2Y2 nucleotide receptor (P2Y2R) regulates the production of LTα. in vitro. We aimed to uncover a potential pathway linking purinergic receptor to LTα-mediated inflammatory processes pivotal to the early stages of atherosclerosis in apolipoprotein E (ApoE−/−) deficient mice. Methods and results En face immunostaining revealed that P2Y2R and VCAM-1 are preferentially expressed in the atherosclerosis prone site of the mouse aortic sinus. Deletion of the P2Y2R gene suppresses VCAM-1 expression. Compared with ApoE−/−mice, ApoE−/−mice lacking the P2Y2R gene (ApoE−/−/P2Y2R−/−) did not develop fatty streak lesions when fed a standard chow diet for 15 weeks. Systemic and CD4+ T cell production of the pro-inflammatory cytokine lymphotoxin-alpha (LTα) were specifically inhibited in ApoE−/−/P2Y2R−/− mice. Anti-LTα preventive treatment was initiated in ApoE−/− mice with intraperitoneal administration of recombinant human tumor necrosis factor receptor 1 fusion protein (TNFR1-Fc) on 5 consecutive days before the disease onset. Remarkably, none of the TNFR1:Fc-treated ApoE−/− mice exhibited atherosclerotic lesions at any developmental stage. Significance ApoE−/− mice deficient in P2Y2R exhibit low endothelial cell VCAM-1 levels, decreased production of LTα and delayed onset of atherosclerosis. These data suggest that targeting this nucleotide receptor could be an effective therapeutic approach in atherosclerosis.

The identity of radical species associated with particulate formed from the oxidative pyrolysis of 1-methylnaphthalene (1-MN) was investigated using low temperature matrix isolation electron paramagnetic resonance spectroscopy (LTMI-EPR), a specialized technique that provided a method of sampling and analysis of the gas-phase paramagnetic components. A superimposed EPR signal was identified to be a mixture of organic radicals (carbon and oxygen-centered) and soot. The carbon-centered radicals were identified as a mixture of the resonance-stabilized indenyl, cyclopentadienyl, and naphthalene 1-methylene radicals through the theoretical simulation of the radical’s hyperfine structure. Formation of these radical species was promoted by the addition of Fe(III)2O3 nanoparticles. Enhanced formation of resonance stabilized radicals from the addition of Fe(III)2O3 nanoparticles can account for the observed increased sooting tendency associated with Fe(III)2O3 nanoparticle addition.

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To identify microRNAs (miRs) that are regulated by Brahmarelated gene 1 (Brg1) in smooth muscle cells (SMCs), we performed microRNA array screens of colonic smooth muscle. Quantitative reverse transcription‐polymerase chain reaction assays confirmed that 6 miRs were down‐regulated and 6 up‐regulated in smooth muscle specific Brg1 knockout tissue. Inactivation of endogenous Brg1 by introducing dominant negative Brg1 into wild type SMCs in vitro, revealed that miR‐143/145 are likely to be directly regulated by Brg1 in smooth muscle cells. miR‐143/145 have been previously shown to be regulated by myocardin and to be important for regulating the phenotype of smooth muscle cells. In Brg1 null SW13 cells, miR‐143/145 were dramatically induced by myocardin only in the presence of Brg1. Chromatin immunoprecipitation assays demonstrated that myocardin increased the binding of serum response factor to the miR‐143/145 promoter only in the presence of Brg1. Together these data suggest a mechanism in which Brg1 containing SWI/SNF complexes are required for myocardin to induce expression of miR‐143/145 in smooth muscle. This suggests that SWI/SNF mediated chromatin remodeling regulates the phenotype of smooth muscle not only by directly affecting expression of smooth muscle specific genes, but also by regulating expression of microRNAs that further modulate expression of their targets.Supported by NIH DK61130.