Sex Differences and Role of Lysyl Oxidase Like 2 (LOXL2) in Angiotensin II-Induced Hypertension in Mice.

BACKGROUND: Hypertension, a disease with known sexual dimorphism, accelerates aging associated arterial stiffening, in part due to the activation of matrix remodeling caused by increased biomechanical load. In this study, we tested the effect of biological sex and the role of the matrix remodeling enzyme lysyl oxidase like 2 (LOXL2) in hypertension induced arterial stiffening. METHODS: Angiotensin II (Ang II) was delivered using osmotic pumps in Loxl2+/- and WT male and female mice. Blood pressure and pulse wave velocity (PWV) were measured noninvasively to assess hypertension and aortic stiffness. Wire myography and uniaxial tensile testing were used to test aortic vasoreactivity and mechanical properties. Aortic wall composition was examined by histology and Western blotting. The effect of biomechanical strain on LOXL2 expression and secretion by vascular smooth muscle (VSMC) and endothelial cells (EC) was evaluated by uniaxial cyclic stretching of cultured cells. The role of LOXL2 catalytic function on VSMC alignment in response to mechanical loading was determined with LOXL2 inhibition and knockout. RESULTS: Ang II infusion induced hypertension in WT and Loxl2+/- mice of both sexes and increased PWV in WT males but not in Loxl2+/- males, WT females, or Loxl2+/- females. LOXL2 depletion protected males from Ang II mediated potentiation of vasoconstriction but worsened in females and improved aortic mechanical properties in both sexes. Histological analysis showed increased aortic wall thickness in hypertensive WT males but not females and increased intralamellar distance in both sexes, that was ameliorated in Loxl2+/- mice. Western blotting revealed increased collagen I, decreased collagen IV, and increased LOXL2 accumulation and processing in hypertensive mice. Hypertensive cyclic strain contributed to LOXL2 upregulation in the cell-derived matrix in VSMCs but not ECs. LOXL2 catalytic function facilitated VSMC alignment in response to biomechanical strain. CONCLUSIONS: In males, arterial stiffening in hypertension is driven both by VSMC response and matrix remodeling. Females exhibit a delayed onset of Ang II-induced hypertension with minimal ECM remodeling but with VSMC dysfunction. LOXL2 depletion ameliorates arterial stiffening and preserves functional contractility and aortic structure in male hypertensive mice. LOXL2 depletion improves aortic mechanics but worsens aortic contractility in hypertensive females. VSMCs are the primary source of LOXL2 in the aorta and hypertension increases LOXL2 processing and shifts to collagen I accumulation. Overall, LOXL2 depletion offers protection in young hypertensive males and females.

LOXL2 inhibition ameliorates pulmonary artery remodeling in pulmonary hypertension.

Background: Conduit pulmonary arterial stiffening and the resultant increase in pulmonary vascular impedance has emerged as an important underlying driver of pulmonary arterial hypertension (PAH). Given that matrix deposition is central to vascular remodeling, we evaluated the role of the collagen crosslinking enzyme lysyl oxidase like 2 (LOXL2) in this study. Methods and Results: Human pulmonary artery smooth muscle cells (PASMCs) subjected to hypoxia showed increased LOXL2 secretion. LOXL2 activity and expression were markedly higher in primary PASMCs isolated from pulmonary arteries of the rat Sugen 5416 + hypoxia (SuHx) model of severe PH. Similarly, LOXL2 protein and mRNA levels were increased in pulmonary arteries (PA) and lungs of rats with PH (SuHx and monocrotaline (MCT) models). Pulmonary arteries (PAs) isolated from rats with PH exhibited hypercontractility to phenylephrine and attenuated vasorelaxation elicited by acetylcholine, indicating severe endothelial dysfunction. Tensile testing revealed a a significant increase in PA stiffness in PH. Treatment with PAT-1251, a novel small-molecule LOXL2 inhibitor, improved active and passive properties of the PA ex vivo. There was an improvement in right heart function as measured by right ventricular pressure volume loops in-vivo with PAT-1251. Importantly PAT-1251 treatment ameliorated PH, resulting in improved pulmonary artery pressures, right ventricular remodeling, and survival. Conclusion: Hypoxia induced LOXL2 activation is a causal mechanism in pulmonary artery stiffening in PH, as well as pulmonary artery mechanical and functional decline. LOXL2 inhibition with PAT-1251 is a promising approach to improve pulmonary artery pressures, right ventricular elastance, cardiac relaxation, and survival in PAH. New & Noteworthy: Pulmonary arterial stiffening contributes to the progression of PAH and the deterioration of right heart function. This study shows that LOXL2 is upregulated in rat models of PH. LOXL2 inhibition halts pulmonary vascular remodeling and improves PA contractility, endothelial function and improves PA pressure, resulting in prolonged survival. Thus, LOXL2 is an important mediator of PA remodeling and stiffening in PH and a promising target to improve PA pressures and survival in PH.

Neonatal exposure to hypoxia induces early arterial stiffening via activation of lysyl oxidases.

Hypoxia in the neonatal period is associated with early manifestations of adverse cardiovascular health in adulthood including higher risk of hypertension and atherosclerosis. We hypothesize that this occurs due to activation of lysyl oxidases (LOXs) and the remodeling of the large conduit vessels, leading to early arterial stiffening. Newborn C57Bl/6 mice were exposed to hypoxia (FiO2  = 11.5%) from postnatal day 1 (P1) to postnatal day 11 (P11), followed by resumption of normoxia. Controls were maintained in normoxia. Using in vivo (pulse wave velocity; PWV) and ex vivo (tensile testing) arterial stiffness indexes, we determined that mice exposed to neonatal hypoxia had significantly higher arterial stiffness compared with normoxia controls by young adulthood (P60), and it increased further by P120. Echocardiography performed at P60 showed that mice exposed to hypoxia displayed a compensated dilated cardiomyopathy. Western blotting revelated that neonatal hypoxia accelerated age-related increase in LOXL2 protein expression in the aorta and elevated LOXL2 expression in the PA at P11 with a delayed decay toward normoxic controls. In the heart and lung, gene and protein expression of LOX/LOXL2 were upregulated at P11, with a delayed decay when compared to normoxic controls. Neonatal hypoxia results in a significant increase in arterial stiffness in early adulthood due to aberrant LOX/LOXL2 expression. This suggests an acceleration in the mechanical decline of the cardiovascular system, that contributes to increased risk of hypertension in young adults exposed to neonatal hypoxia that may increase susceptibility to further insults.

Off-the-shelf, heparinized small diameter vascular graft limits acute thrombogenicity in a porcine model.

Thrombogenicity poses a challenge to the clinical translation of engineered grafts. Previously, small-diameter vascular grafts (sdVG) composed of fibrin hydrogel microfiber tubes (FMT) with an external poly(ε-caprolactone) (PCL) sheath supported long-term patency in mice. Towards the development of an sdVG with off-the-shelf availability, the FMT's shelf stability, scale-up, and successful conjugation of an antithrombotic drug to the fibrin scaffold are reported here. FMTs maintain mechanical stability and high-water retention after storage for one year in a freezer, in a refrigerator, or at room temperature. Low molecular weight heparin-conjugated fibrin scaffolds enabled local and sustained delivery during two weeks of enzymatic degradation. Upscaled fabrication of sdVGs provides natural biodegradable grafts with size and mechanics suitable for human application. Implantation in a carotid artery interposition porcine model exhibited no rupture with thrombi prevented in all heparinized sdVGs (n = 4) over 4-5 weeks. Remodeling of the sdVGs is demonstrated with endothelial cells on the luminal surface and initial formation of the medial layer by 4-5 weeks. However, neointimal hyperplasia at 4-5 weeks led to the stenosis and occlusion of most of the sdVGs, which must be resolved for future long-term in vivo assessments. The off-the-shelf, biodegradable heparinized fibrin sdVG layer limits acute thrombogenicity while mediating extensive neotissue formation as the PCL sheath maintains structural integrity. STATEMENT OF SIGNIFICANCE: To achieve clinical and commercial utility of small-diameter vascular grafts as arterial conduits, these devices must have off-the-shelf availability for emergency arterial bypass applications and be scaled to a size suitable for human applications. A serious impediment to clinical translation is thrombogenicity. Treatments have focused on long-term systemic drug therapy, which increases the patient's risk of bleeding complications, or coating grafts and stents with anti-coagulants, which minimally improves patient outcomes even when combined with dual anti-platelet therapy. We systematically modified the biomaterial properties to develop anticoagulant embedded, biodegradable grafts that maintain off-the-shelf availability, provide mechanical stability, and prevent clot formation through local drug delivery.

Integrating morphology and in vivo skeletal mobility with digital models to infer function in brittle star arms.

Brittle stars (Phylum Echinodermata, Class Ophiuroidea) have evolved rapid locomotion employing muscle and skeletal elements within their (usually) five arms to apply forces in a manner analogous to that of vertebrates. Inferring the inner workings of the arm has been difficult as the skeleton is internal and many of the ossicles are sub-millimeter in size. Advances in 3D visualization and technology have made the study of movement in ophiuroids possible. We developed six virtual 3D skeletal models to demonstrate the potential range of motion of the main arm ossicles, known as vertebrae, and six virtual 3D skeletal models of non-vertebral ossicles. These models revealed the joint center and relative position of the arm ossicles during near-maximal range of motion. The models also provide a platform for the comparative evaluation of functional capabilities between disparate ophiuroid arm morphologies. We made observations on specimens of Ophioderma brevispina and Ophiothrix angulata. As these two taxa exemplify two major morphological categories of ophiuroid vertebrae, they provide a basis for an initial assessment of the functional consequences of these disparate vertebral morphologies. These models suggest potential differences in the structure of the intervertebral articulations in these two species, implying disparities in arm flexion mechanics. We also evaluated the differences in the range of motion between segments in the proximal and distal halves of the arm length in a specimen of O. brevispina, and found that the morphology of vertebrae in the distal portion of the arm allows for higher mobility than in the proximal portion. Our models of non-vertebral ossicles show that they rotate further in the direction of movement than the vertebrae themselves in order to accommodate arm flexion. These findings raise doubts over previous hypotheses regarding the functional consequences of ophiuroid arm disparity. Our study demonstrates the value of integrating experimental data and visualization of articulated structures when making functional interpretations instead of relying on observations of vertebral or segmental morphology alone. This methodological framework can be applied to other ophiuroid taxa to enable comparative functional analyses. It will also facilitate biomechanical analyses of other invertebrate groups to illuminate how appendage or locomotor function evolved.