Increased arterial stiffness is an independent risk factor for hypertension, stroke, and cardiovascular morbidity. Thus, understanding the factors contributing to vascular stiffness is of critical importance. Here, we used a rat model containing a known quantitative trait locus (QTL) on chromosome 3 (RNO3) for vasoreactivity to assess potential genetic elements contributing to blood pressure, arterial stiffness, and their downstream effects on cardiac structure and function. Although no differences were found in blood pressure at any time point between parental spontaneously hypertensive rats (SHRs) and congenic SHR.BN3 rats, the SHRs showed a significant increase in arterial stiffness measured by pulse wave velocity. The degree of arterial stiffness increased with age in the SHRs and was associated with compensatory cardiac changes at 16 wk of age, and decompensatory changes at 32 wk, with no change in cardiac structure or function in the SHR.BN3 hearts at these time points. To evaluate the arterial wall structure, we used multiphoton microscopy to quantify cells and collagen content within the adventitia and media of SHR and SHR.BN3 arteries. No difference in cell numbers or proliferation rates was found, although phenotypic diversity was characterized in vascular smooth muscle cells. Herein, significant anatomical and physiological differences related to arterial structure and cardiovascular tone including collagen, pulse wave velocity (PWV), left ventricular (LV) geometry and function, and vascular smooth muscle cell (VSMC) contractile apparatus proteins were associated with the RNO3 QTL, thus providing a novel platform for studying arterial stiffness. Future studies delimiting the RNO3 QTL could aid in identifying genetic elements responsible for arterial structure and function.

Impaired liver regeneration has been considered as a hallmark of progression of alcohol-associated liver disease. Our previous studies demonstrated that in vivo inhibition of the microRNA (miRNA) miR21 can restore regenerative capacity of the liver in chronic ethanol-fed animals. The present study focuses on the role of microRNA regulatory networks that are likely to mediate the miR-21 action. Rats were chronically fed an ethanol-enriched diet along with pair-fed control animals and treated with AM21 (anti-miR-21), a locked nucleic acid antisense to miR-21. Partial hepatectomy (PHx) was performed and miRNA expression profiling over the course of liver regeneration was assessed. Our results showed dynamic expression changes in several miRNAs after PHx, notably with altered miRNA expression profiles between ethanol and control groups. We found that in vivo inhibition of miR-21 led to correlated differential expression of miR-340-5p and anticorrelated expression of miR-365, let-7a, miR-1224, and miR-146a across all sample groups after PHx. Gene set enrichment analysis identified a miRNA signature significantly associated with hepatic stellate cell activation within whole liver tissue data. We hypothesized that at least part of the PHx-induced miRNA network changes responsive to miR-21 inhibition is localized to hepatic stellate cells. We validated this hypothesis using AM21 and TGF-β treatments in LX-2 human hepatic stellate cells in culture and measured expression levels of select miRNAs by quantitative RT-PCR. Based on the in vivo and in vitro results, we propose a hepatic stellate cell miRNA regulatory network as contributing to the restoration of liver regenerative capacity by miR-21 inhibition.

Integration of microbiota in a host begins at birth and progresses during adolescence, forming a multidirectional system of physiological interactions. Here, we present an instantaneous effect of natural, bacterial gut colonization on the acceleration of longitudinal and radial bone growth in germ-free born, 7-wk-old male rats. Changes in bone mass and structure were analyzed after 10 days following the onset of colonization through cohousing with conventional rats and revealed unprecedented acceleration of bone accrual in cortical and trabecular compartments, increased bone tissue mineral density, improved proliferation and hypertrophy of growth plate chondrocytes, bone lengthening, and preferential deposition of periosteal bone in the tibia diaphysis. In addition, the number of small in size adipocytes increased, whereas the number of megakaryocytes decreased, in the bone marrow of conventionalized germ-free rats indicating that not only bone mass but also bone marrow environment is under control of gut microbiota signaling. The changes in bone status paralleled with a positive shift in microbiota composition toward short-chain fatty acids (SCFA)-producing microbes and a considerable increase in cecal SCFA concentrations, specifically butyrate. Furthermore, reconstitution of the host holobiont increased hepatic expression of IGF-1 and its circulating levels. Elevated serum levels of 25-hydroxy vitamin D and alkaline phosphatase pointed toward an active process of bone formation. The acute stimulatory effect on bone growth occurred independently of body mass increase. Overall, the presented model of conventionalized germ-free rats could be used to study microbiota-based therapeutics for combatting dysbiosis-related bone disorders.