Coronary heart disease is a leading cause of death among Americans for which coronary artery bypass graft (CABG) surgery is a standard surgical treatment. The success of CABG surgery is impaired by a compliance mismatch between vascular grafts and native vessels. Tissue engineered vascular grafts (TEVGs) have the potential to be compliance matched and thereby reduce the risk of graft failure. Glutaraldehyde (GLUT) vapor-crosslinked gelatin/fibrinogen constructs were fabricated and mechanically tested in a previous study by our research group at 2, 8, and 24 hrs of GLUT vapor exposure. The current study details a computational method that was developed to predict the material properties of our constructs for crosslinking times between 2 and 24 hrs by interpolating the 2, 8, and 24 hrs crosslinking time data. matlab and abaqus were used to determine the optimal combination of fabrication parameters to produce a compliance matched construct. The validity of the method was tested by creating a 16-hr crosslinked construct of 130 μm thickness and comparing its compliance to that predicted by the optimization algorithm. The predicted compliance of the 16-hr construct was 0.00059 mm Hg-1 while the experimentally determined compliance was 0.00065 mm Hg-1, a relative difference of 9.2%. Prior data in our laboratory has shown the compliance of the left anterior descending porcine coronary (LADC) artery to be 0.00071 ± 0.0003 mm Hg-1. Our optimization algorithm predicts that a 258-μm-thick construct that is GLUT vapor crosslinked for 8.1 hrs would match LADC compliance. This result is consistent with our previous work demonstrating that an 8-hr GLUT vapor crosslinked construct produces a compliance that is not significantly different from a porcine coronary LADC.

T cells are required for the development of hypertension in male and postmenopausal female mice while premenopausal females are protected from T cell mediated hypertension. To better understand sex differences in immune-cell mediated hypertensive responses, we sought to determine if there were any significant differences in the immune cell profiles of premenopausal female (F), VCD-treated postmenopausal female (PMF), and male (M) mice. Spleens were collected from all mice and processed for flow cytometric analysis of T cell populations (n=8/group). Analysis of splenic T cell populations revealed no significant difference in the frequency of CD3+ or CD4+ T cells between groups (CD3+: F 33.4%, PMF 30.3%, M 30.2% of total lymphocytes, CD4+: F 64.8%, PMF 70.7%, M 67.5% of CD3+ cells). However, postmenopausal females had a significantly lower frequency of splenic CD8+ T cells compared to both males and premenopausal females (CD8+: F 27.9%, PMF 19.5%*, M 25.3% of CD3+ cells *p<0.05 vs M and F). Additionally, premenopausal females displayed significantly increased expression of the memory marker CD44 and the anti-inflammatory marker CTLA-4 on CD4+ cells compared to both males and postmenopausal females (MFI CD44: F 334.8*, PMF 269.4, M 280.4, MFI CTLA-4: F 100.7*, PMF 80.9, M 86.8 *p<0.01 vs M and PMF). Additional flow cytometric staining was performed to evaluate sex differences in splenic Antigen Presenting Cell (APC) populations (n=8/group). The frequency of CD11b+ APCs, thought to primarily represent macrophage populations, were significantly reduced in postmenopausal females compared to premenopausal females but there was no significant difference from males (CD11b +: F 14.6%, PMF 11.8%*, M 12.9% of monocytes *p<0.05 vs F). Additionally, CD11c+ dendritic cell populations were found to be significantly reduced in postmenopausal females compared to both males and premenopausal females (CD11c+: F 4.1%, PMF 2.8%*, M 3.9% of all monocytes *p<0.01 vs M and F). Taken together, these results indicate a significant difference in the baseline immune environment between male, premenopausal female and postmenopausal females which likely contribute to sex-differences in susceptibility to immune-mediated hypertension.

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There is extensive evidence that the immune system is required for the development of angiotensin II (Ang II) induced hypertension in males. In contrast, before entering menopause, we have shown that females are protected from T cell‐mediated Ang II hypertension. Following menopause, female protection from T cell‐mediated Ang II hypertension is lost; in the absence of T cells, systolic blood pressure (SBP) responses to Ang II in Rag‐1−/− postmenopausal mice were similar to those seen in premenopausal mice (SBP Δ12 ± 2 mmHg). After adoptive transfer of CD3+ T cells, Ang II significantly increased SBP in postmenopausal females (SBP Δ28 ± 3 mmHg; p<0.05). Thus, we hypothesize that the loss of estrogen in postmenopausal mice eliminates female protection to T cell‐mediated hypertension. Further, we examined if premenopausal protection from T cell‐mediated hypertension was due to T cell‐specific estrogen receptor alpha (ERα) signaling. CD3+ T cells were purified from ERα null mice (ERαKO) and adoptively transferred into cycling, female Rag‐1−/− mice (no T/B cells). Ang II was infused via osmotic mini‐pump for 14 days (490 ng/kg/min). Similar to our previous studies, cycling female Rag‐1−/− mice were resistant to T cell‐mediated Ang II hypertension following T cell transfer from wild type mice (SBP CD3+WT/Ang Δ6 ± 5 mmHg). Additionally, there was no significant difference in SBP when ERαKO T cells were adoptively transferred (SBP CD3+ ERαKO/Ang Δ5 ± 4 mmHg). Flow cytometric analysis of renal CD4+, CD8+, and regulatory Foxp3+ T cells identified that in the absence of ERα, there was an increase in expression of costimulatory receptor CD28 in all three T cell subsets (CD3+ ERαKO/Ang vs. CD3+WT/Ang: CD4+ 138%, CD8+ 144%, Foxp3+ 141%; p<0.05). Our studies suggest that loss of estrogen (menopause) increases female susceptibility to T cell‐mediated hypertension. However, loss of T cell‐specific ERα signaling does not seem to play a key role in premenopausal protection from T cell‐mediated Ang II hypertension.Support or Funding Information1. T32HL0072492. R01HL131834This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cognitive decline, as seen in Alzheimer’s disease (AD), is a growing public health concern and is linked to decreased cerebral blood flow, particularly in women after menopause. Impaired cerebrovascular function precedes the onset of AD, possibly due to reduced endothelial function in parenchymal arterioles, the bottlenecks of the cerebral microcirculation, although exact mechanisms by menopause remain elusive. The goal of this study was to determine whether menopause impairs endothelial function in parenchymal arterioles of wild‐type (WT) and 5x‐FAD mouse model of AD. Menopause was induced by the 4‐vinylcyclohexene diepoxide (VCD) model, which induces gradual ovarian failure. Cerebral parenchymal arterioles were isolated and studied by pressure myography. Data are means ± SEM. In WT mice, menopause caused a significant increase in myogenic tone (myogenic tone: 24.73 ± 1.2 vs. 34 ± 2.6%, vehicle vs VCD, n = 5 / 8; p<0.05), as well as a small but significant reduction in resting lumen diameter (32.68 ± 1.3 vs. 28.40 ± 2.1 µm, vehicle vs VCD, n = 5 / 8; p<0.05). We then tested endothelial function, focusing on K+ channels, namely small and intermediate Ca2+‐activated K+ channels (SKCa / IKCa) and inwardly rectifying K+ channels (KIR2). Menopause did not affect arteriolar dilation to a cumulative concentration‐response curve of the SKCa/ IKCa activator NS‐309 (at 1 µM, vasodilation: 23.49 ± 11.5 vs. 17.80 ± 1.7%, vehicle vs VCD, n = 3 / 6). Similarly, KIR2 function was unchanged (vasodilation: 16.06 ± 6.6 vs. 16.21 ± 2.9%, vehicle vs VCD, n = 4 / 8). In the 5x‐FAD mice, menopause did not affect myogenic tone (myogenic tone: 23.49 ± 2.2 vs. 28 3.2%, vehicle vs VCD, n = 10 / 7), although there was a significant decrease in resting lumen diameter (39.16 ± 2.6 vs. 27.79 ± 2.0 µm, vehicle vs VCD, n = 10 / 7 p<0.05). Arterioles from menopausal 5x‐FAD mice showed a significantly blunted response to NS‐309 (at 1 µM, vasodilation: 14.96 ± 2.9 vs. 9.42 ± 1.4%, vehicle vs VCD, n = 7 / 4, p<0.05), without differences in KIR2‐induced dilation (vasodilation: 16.02 ± 1.3 vs. 13.69 ± 2.3%, vehicle vs VCD n = 9 / 6). In conclusion, we found that in WT mice, menopause leads to changes in myogenic tone without affecting K+‐dependent dilation. However, in 5x‐FAD mice, menopause impairs endothelial SKCa/IKCa channels independent of KIR2 function or myogenic tone. These findings identify new targets modulated by menopause in the cerebral microvasculature of WT and AD mice, which may contribute to cognitive dysfunction.