Echocardiographic markers of pulmonary hemodynamics and right ventricular hypertrophy in rat models of pulmonary hypertension.

Echocardiography is the gold standard non-invasive technique to diagnose pulmonary hypertension. It is also an important modality used to monitor disease progression and response to treatment in patients with pulmonary hypertension. Surprisingly, only few studies have been conducted to validate and standardize echocardiographic parameters in experimental animal models of pulmonary hypertension. We sought to define cut-off values for both invasive and non-invasive measures of pulmonary hemodynamics and right ventricular hypertrophy that would reliably diagnose pulmonary hypertension in three different rat models. The study was designed in two phases: (1) a derivation phase to establish the cut-off values for invasive measures of right ventricular systolic pressure, Fulton's index (right ventricular weight/left ventricle + septum weight), right ventricular to body weight ratio, and non-invasive echocardiographic measures of pulmonary arterial acceleration time, pulmonary arterial acceleration time to ejection time ratio and right ventricular wall thickness in diastole in the hypoxic and monocrotaline rat models of pulmonary hypertension and (2) a validation phase to test the performance of the cut-off values in predicting pulmonary hypertension in an independent cohort of rats with Sugen/hypoxia-induced pulmonary hypertension. Our study demonstrates that right ventricular systolic pressure ≥35.5 mmHg and Fulton's Index ≥0.34 are highly sensitive (>94%) and specific (>91%) cut-offs to distinguish animals with pulmonary hypertension from controls. When pulmonary arterial acceleration time/ejection time and right ventricular wall thickness in diastole were both measured, a result of either pulmonary arterial acceleration time/ejection time ≤0.25 or right ventricular wall thickness in diastole ≥1.03 mm detected right ventricular systolic pressure ≥35.5 mmHg or Fulton's Index ≥0.34 with a sensitivity of 88% and specificity of 100%. With properly validated non-invasive echocardiography measures of right ventricular performance in rats that accurately predict invasive measures of pulmonary hemodynamics, future studies can now utilize these markers to test the efficacy of different treatments with preclinical therapeutic modeling.

Heme oxygenase-1 dampens the macrophage sterile inflammasome response and regulates its components in the hypoxic lung.

Exposure to hypoxia causes an inflammatory reaction in the mouse lung, and this response can be modulated by overexpressing the hypoxia-inducible stress-response enzyme, heme oxygenase-1 (HO-1). We hypothesized that the inflammasome activity may be a central pathway by which HO-1 controls pulmonary inflammation following alveolar hypoxia. Therefore, we investigated whether HO-1 controls inflammasome activation by altering its expression in macrophages primed with classic NOD-like receptor containing a pyrin domain 3 (NLRP3) inducers, and in murine lungs lacking HO-1 and exposed to acute hypoxia. We found that lack of HO-1 activated lipopolysaccharide (LPS) and ATP-treated bone marrow-derived macrophages, causing an increase in secreted levels of cleaved interleukin (IL)-1B, IL-18, and caspase-1, markers of increased inflammasome activity, whereas HO-1 overexpression suppressed IL-1B, NLRP3, and IL-18. The production of cleaved IL-1B and the activation of caspase-1 in LPS- and ATP-primed macrophages were inhibited by hemin, an HO-1 inducer, and two HO-1 enzymatic products [bilirubin and carbon monoxide (CO)]. Exposure of mice to hypoxia induced the expression of several inflammasome mRNA components (IL-1B, Nlrp3, and caspase-1), and this was further augmented by HO-1 deficiency. This pronounced inflammasome activation was detected as increased protein levels of apoptosis-associated speck-like protein containing a COOH-terminal caspase recruitment domain, IL-18, procaspase-1, and cleaved caspase-1 in the lungs of hypoxic mice. Systemically, Hmox1-deficient mice showed increased basal levels of IL-18 that were further increased after 48 h of hypoxic exposure. Taken together, these finding point to a pivotal role for HO-1 in the control of baseline and hypoxic inflammasome signaling, perhaps through the antioxidant properties of bilirubin and CO's pleiotropic effects.

Distal vessel stiffening is an early and pivotal mechanobiological regulator of vascular remodeling and pulmonary hypertension.

Pulmonary arterial (PA) stiffness is associated with increased mortality in patients with pulmonary hypertension (PH); however, the role of PA stiffening in the pathogenesis of PH remains elusive. Here, we show that distal vascular matrix stiffening is an early mechanobiological regulator of experimental PH. We identify cyclooxygenase-2 (COX-2) suppression and corresponding reduction in prostaglandin production as pivotal regulators of stiffness-dependent vascular cell activation. Atomic force microscopy microindentation demonstrated early PA stiffening in experimental PH and human lung tissue. Pulmonary artery smooth muscle cells (PASMC) grown on substrates with the stiffness of remodeled PAs showed increased proliferation, decreased apoptosis, exaggerated contraction, enhanced matrix deposition, and reduced COX-2-derived prostanoid production compared with cells grown on substrates approximating normal PA stiffness. Treatment with a prostaglandin I2 analog abrogated monocrotaline-induced PA stiffening and attenuated stiffness-dependent increases in proliferation, matrix deposition, and contraction in PASMC. Our results suggest a pivotal role for early PA stiffening in PH and demonstrate the therapeutic potential of interrupting mechanobiological feedback amplification of vascular remodeling in experimental PH.

The Sugen 5416/hypoxia mouse model of pulmonary hypertension revisited: long-term follow-up.

The combination of a vascular endothelial growth factor receptor antagonist, Sugen 5416 (SU5416), and chronic hypoxia is known to cause pronounced pulmonary hypertension (PH) with angioobliterative lesions in rats and leads to exaggerated PH in mice as well. We sought to determine whether weekly SU5416 injections during 3 weeks of hypoxia leads to long-term development of angioobliterative lesions and sustained or progressive PH in mice. Male C57BL/6J mice were injected with SU5416 (SuHx) or vehicle (VehHx) weekly during 3 weeks of exposure to 10% oxygen. Echocardiographic and invasive measures of hemodynamics and pulmonary vascular morphometry were performed after the 3-week hypoxic exposure and after 10 weeks of recovery in normoxia. SuHx led to higher right ventricular (RV) systolic pressure and RV hypertrophy than VehHx after 3 weeks of hypoxia. Ten weeks after hypoxic exposure, RV systolic pressure decreased but remained elevated in SuHx mice compared with VehHx or normoxic control mice, but RV hypertrophy had resolved. After 3 weeks of hypoxia and 10 weeks of follow-up in normoxia, tricuspid annular plane systolic excursion was significantly decreased, indicating decreased systolic RV function. Very few angioobliterative lesions were found at the 10-week follow-up time point in SuHx mouse lungs. In conclusion, SU5416 combined with 3 weeks of hypoxia causes a more profound PH phenotype in mice than hypoxia alone. PH persists over 10 weeks of normoxic follow-up in SuHx mice, but significant angioobliterative lesions do not occur, and neither PH nor RV dysfunction worsens. The SuHx mouse model is a useful adjunct to other PH models, but the search will continue for a mouse model that better recapitulates the human phenotype.