MicroRNAs (miRNAs) may regulate a number of genes, each of which may have a variety of functions. We utilized an endoarterial biopsy catheter to assess the dysregulation of miRNAs in a porcine shunt model of pulmonary hypertension (PH). Two Yucatan micropigs underwent surgical anastomosis of the left pulmonary artery to the descending aorta. Endoarterial biopsy samples were obtained at baseline, and at regular intervals during the progression of PH. RNA, isolated from biopsy samples, was analyzed by Illumina miRNA expression microarrays (containing ∼1200 human miRNAs), Affymetrix Porcine GeneChips, Bioconductor, and GeneSpring. We examined a total of 925 genes in a PH whole genome microarray. Biopsy samples showed that 39 miRNAs were downregulated and 34 miRNAs were upregulated compared to baseline. The number of PH-associated genes reported to be controlled by each of the dysregulated miRNAs was in the range of 1–113. The five miRNAs that had the largest number of PH-associated genes were: miR-548c-3p, miR-520d-3p, miR-130a-5p, miR-30a-3p, and miR-let-7g-3p. Several of the dysregulated miRNAs have been associated with molecular pathways and biologic processes involved in PH. Among 29 miRNAs, which were predicted to be dysregulated by a systems biology approach, we found four that were dysregulated in our porcine shunt model. An endoarterial biopsy technique was successful in showing that a large number of miRNAs are dysregulated in a porcine shunt model of PH. Many of these miRNAs control multiple PH-associated genes, molecular pathways, and biologic processes. Endoarterial biopsy offers potential experimental and clinical diagnostic value.

The molecular mechanisms of pulmonary arterial hypertension (PAH) remain ill-defined. The aims of this study were to obtain sequential endoarterial biopsy samples in a surgical porcine model of PAH and assess changes in histology and mRNA expression during the disease progression. Differentially expressed genes were then analyzed as potential pharmacological targets. Four Yucatan micro-pigs underwent surgical anastomosis of the left pulmonary artery to the descending aorta. Endovascular samples were obtained with a biopsy catheter at baseline (before surgery) and from the left lung 7, 60, and 180 days after surgery. RNA was isolated from biopsy samples, amplified and analyzed. Dysregulated genes were linked to drugs with potential to treat or prevent PAH. With the development of PAH in our model, we identified changes in histology and in the expression of several genes with known or investigational inhibitors and several novel genes for PAH. Gene dysregulation displayed time-related variations during disease progression. Endoarterial biopsy provides a new method of assessing pulmonary vascular histology and gene expression in PAH. This analysis could identify novel applications for existing and new PAH drugs. The detection of stage- and disease-specific variation in gene expression could lead to individualized therapies.

Pulmonary hypertension (PH) results in significant morbidity and mortality. Chronic PH animal models may advance the study of PH’s mechanisms, evolution, and therapy. In this report, we describe the challenges and successes in developing three models of chronic PH in large animals: two models (one canine and one swine) utilized repeated infusions of ceramic microspheres into the pulmonary vascular bed, and the third model employed a surgical aorto-pulmonary shunt. In the canine model, seven dogs underwent microsphere infusions that resulted in progressive elevation of pulmonary arterial pressure over a few months. In this model, pulmonary endoarterial tissue was obtained for histology. In the aorto-pulmonary shunt swine model, 17 pigs developed systemic level pulmonary pressures after 2–3 months. In this model, pulmonary endoarterial tissue was sequentially obtained to assess for changes in gene and microRNA expression. In the swine microsphere infusion model, three pigs developed only a modest chronic increase in pulmonary arterial pressure, despite repeated infusions of microspheres (up to 40 in one animal). The main purpose of this model was for vasodilator testing, which was performed successfully immediately after acute microsphere infusions. Chronic PH in large animal models can be successfully created; however, a model’s characteristics need to match the investigational goals.

Disuse atrophy of both muscle and bone can occur rapidly during periods of inactivity. In several rodent models developed for the study of disuse atrophy, immobilization is induced by prolonged cage restraint, hind limb unloading, tenotomy, sciatic nerve block or sciatic denervation. In less tractable species such as wild-caught hibernating rodents, the sciatic denervation model is superior in terms of both animal welfare and applicability to the characteristics of natural cases of disuse atrophy. The authors describe a refined surgical approach to sciatic denervation in golden-mantled ground squirrels (Callospermophilus lateralis), a hibernating species, that improves animal welfare and reduces the incidence of post-operative complications such as autotomy.

Background: Despite the approval of several medications for pulmonary hypertension, morbidity and mortality are unacceptably high. Systemic hypotension may limit the use of pulmonary hypertension medications. Objectives: This study aimed to assess whether the homing peptide CAR (CARSKNKDC) improves the vasodilatory selectivity of fasudil in the pulmonary circulation or systemic circulation in a porcine pulmonary hypertension model. Materials and Methods: Pulmonary hypertension (to approximately 2/3-3/4 systemic pressure levels) was induced by chronic and acute administration of microspheres in 3 micro Yucatan pigs (mean weight 19.9 kg, mean age 4.3 months). Fasudil (0.3 mg/kg) was administered without and with CAR (1.5 mg/kg), and the effect on aortic (Ao) and right ventricular (RV) pressure was recorded with indwelling catheters. Results: Immediately after fasudil administration, there was a decrease in Ao pressure followed by prompt recovery to baseline. The RV pressure decrease was progressive and sustained. Fasudil alone resulted in a 12% decrease in RV pressure, whereas co-administration of CAR with fasudil resulted in a 22% decrease in RV pressure (p < 0.0001). Fasudil alone caused an average decrease of 34% in the RV/Ao pressure ratio, and fasudil + CAR caused an average decrease of 40% in the RV/Ao pressure ratio (p < 0.0001). Conclusion: The homing peptide CAR selectively enhanced the acute vasodilatory effects of fasudil on the pulmonary vascular bed in a porcine experimental model of pulmonary hypertension.

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Introduction and Objectives. Despite the approval of several medications for pulmonary hypertension, morbidity and mortality are unacceptably high. Systemic hypotension may limit the use of pulmonary hypertension medications. The aim of this study was to assess whether the homing peptide CAR (CARSKNKDC) improved the vasodilatory selectivity of fasudil on the pulmonary circulation vs the systemic circulation in a porcine pulmonary hypertension model. Materials and Methods. Pulmonary hypertension (to approximately 2/3-3/4 systemic pressure levels) was induced by chronic and acute administration of microspheres in 3 micro Yucatan pigs (mean weight 19.9 kg, mean age 4.3 months). Fasudil (0.3 mg/kg) was administered without and with CAR (1.5 mg/kg) and the effects on aortic (Ao) and right ventricular (RV) pressure was recorded with indwelling catheters. Results. Immediately after fasudil administration, there was a decrease in Ao pressure followed by prompt recovery to baseline. The RV pressure decrease was progressive and sustained. Fasudil alone resulted in a 12 % decrease in RV pressure, whereas the co-administration of CAR with fasudil resulted in a 22 % decrease in RV pressure (p < 0.0001). Fasudil alone caused an average decrease of 34% in the RV/Ao pressure ratio and fasudil + CAR caused an average decrease of 40% in the RV/Ao pressure ratio (p < 0.0001). Conclusion. The homing peptide CAR selectively enhances the acute vasodilatory effects of fasudil on the pulmonary vascular bed in a porcine experimental model of pulmonary hypertension.

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