Thoracic aortic aneurysm is one of the manifestations of Marfan syndrome (MFS) that is known to affect men more severely than women. However, the incidence of MFS is similar between men and women. The aim of this study is to show that during pathological aortic dilation, sex-dependent severity of thoracic aortopathy in a mouse model of Marfan syndrome translates into sex-dependent alterations in cells and matrix of the ascending aorta, consequently affecting aortic biomechanics. Fibrillin1 C1041G/+ were used as a mouse model of MFS. Ultrasound measurements from 3-12 months showed increased aortic diameter in Marfan aorta with larger percent increase in diameter for males compared to females. Immunohistochemistry showed decreased contractile smooth muscle cells in Marfan aortic wall compared to healthy aorta, which was accompanied by decreased contractility measured by wire myography. Elastin autofluorescence, second harmonic generation microscopy of collagen fibers and passive biomechanical assessments using myography showed more severe damage to elastin fibers, increased medial fibrosis, and increased stiffness of the aortic wall in MFS males but not females. Male and female heterozygotes showed increased expression of Sca-1-positive adventitial progenitor cells vs. controls at young ages. In agreement with clinical data, Marfan mice demonstrate sex-dependent severity of thoracic aortopathy. It was also shown that aging exacerbates the disease state especially for males. Our findings suggest that female mice are protected from progression of aortic dilation at early ages, leading to a lag in aneurysm growth.

The following major observations were made in this study: 1) aortic adventitial collagen fibers become more longitudinally oriented with aging in apolipoprotein E knockout mice fed a chow diet; 2) conversely, adventitial collagen fibers become more circumferentially oriented with aging in apoE knockout mice fed a high-fat diet; 3) adventitial collagen content increases significantly with age in mice on a high-fat diet; 4) these alterations in collagen organization occur largely in the absence of hemodynamic changes; and 5) circumferential reorientation of collagen is associated with decreased active force generation (contractility) in aged mice on a high-fat diet.

Objectives: Thoracic aortic aneurysm (TAA) is a chronic vascular disease and the 15th leading cause of death in the US. TAA can result from a genetic disorder such as Marfan syndrome (MFS). MFS patients typically have fibrillin-1 deficiency, causing elastic fiber defects in the aortic media that initiate a series of cellular events. We sought to determine the association of smooth muscle cell (SMC) conversion from contractile to synthetic phenotype with changes in extracellular matrix (ECM), medial cell density, and arterial biomechanics. Methods: A mouse model of MFS (Fbn1 C1039G/+, Het) was used to study progression of TAA and cellular events over a 3-12 month time course. Small animal ultrasound was used to image the ascending aorta and to measure diameters at several positions. Immunohistochemistry (IHC) was performed on proximal aorta sections with smooth muscle-myosin heavy chain (SM-MHC) and SM alpha actin as markers for contractile SMCs. Second harmonic generation (SHG) microscopy was used to quantitate aortic medial collagen. Results: Ultrasound data show that aortic diameter significantly increases with time in Het mice. There is a statistically significant difference between genotypes independent of time. IHC images demonstrate that while SM-MHC expression in wild type (WT) mice remains nearly constant, in Het mice at 3 months the expression is comparable to that of WT mice but decreases at 6 months and starts to recover to the WT level by 12 months. SHG images show a time dependent increase in medial collagen expression in Het mice vs. WT. These results suggest loss of contractile SMCs in the media and remodeling of ECM. Conclusion: Our results to date demonstrate loss of contractile SMCs and increased medial collagen deposition in the proximal aorta during TAA progression. Loss of contractile cells together with changes in ECM are expected to alter aortic biomechanics. To quantify these alterations, we are currently studying active and passive biomechanics of ascending aortas of WT and Het mice.

Introduction: Type 2 diabetes mellitus (T2DM)-associated cardiac fibrosis contributes to heart failure, especially in women. We have data showing that diabetic mice with cardiomyopathy, including cardiac fibrosis, exhibit low levels of the neuropeptide substance P (SP); and SP replacement reverses cardiac fibrosis, independent of body weight and blood glucose. To understand the potential translational significance of these findings, we sought to determine in a T2DM monkey model if replacement SP could reverse cardiac magnetic resonance (CMR) imaging and circulating markers of fibrosis, while also assessing safety of administration. Methods: Female T2DM African Green monkeys were randomized to receive SP (n=4, 0.5 mg/Kg/day S.Q. injection) or vehicle (n=3) for 10 weeks. We obtained CMR imaging and blood samples to assess left ventricular (LV) function and fibrosis by T1 map-derived extracellular volume (ECV) and circulating procollagen type I C-terminal propeptide (PICP). Hematological parameters for toxicities were also assessed. We performed a blinded paired analysis of all parameters. Results: Monkeys receiving replacement SP exhibited a decrease in ECV (Pre: 31.6 ± 5.53% vs. Post: 25.2 ± 3.3%, p=0.03), concomitant with decreased PICP levels (Pre: 5148.58 ± 1143.5 ng/mL vs. Post: 3851.1 ± 1377.5 ng/mL, p=0.01). Animals receiving vehicle showed no changes in ECV (p=0.32) or PICP (p=0.16). No changes in LV ejection fraction were observed (p=0.89). Complete blood counts, metabolic panel (ΔA1c, SP: -0.45 ± 0.59 % vs vehicle: -0.53 ± 0.61 %, p=0.43), lipids, liver and pancreatic enzymes, and inflammation markers were unchanged (p>0.05 for all). Conclusion: Replacement SP reversed cardiac fibrosis in a large preclinical model of T2DM, independent of glycemic control. No organ-related toxicity was associated with replacement SP. These results strongly support a potential application for replacement SP as an antifibrotic therapy for diabetic cardiac fibrosis.