The purpose of this study was to define a lower standard cuff occlusion time to induce reactive hyperemia in assessing endothelial dysfunction. In this study, strong evidence was found by a novel technique that used oscillometric methods, which supported that 3 minute reactive hyperemia was sufficient to elicit a significant difference in arterial compliance from baseline. Twenty healthy Cal Poly students were assessed, (n=12 female, n=8 male) aged 22 years old with a standard deviation of 2.04 years. Arterial compliance was estimated by measuring the peak-to-peak oscillations for baseline, 3 minute reactive hyperemia, and 5 minute reactive hyperemia tests, with the result being statistical evidence of an increase in arterial compliance after 3 minutes of cuff occlusion compared to baseline. The peak-to-peak mean for the 3 minute reactive hyperemia test was significantly greater than the baseline peak-to-peak mean with p-values less than 0.0001. These results support that 3 minute reactive hyperemia is sufficient to assess endothelial dysfunction using oscillometry techniques. Endothelial dysfunction is the most significant predictor of a major adverse cardiovascular event, so this test can be used as an early detection tool for cardiovascular disease and allow patients to find treatment before irreversible damage is done to the body. Implementing this test into routine doctor checkups has the potential to have a significant effect on cardiovascular disease, which is the leading cause of death globally. The currently accepted clinical benchmark performed in hospitals uses high-frequency ultrasound with a standard cuff occlusion time of 5 minutes. Although noninvasive, 5 minutes of cuff occlusion causes slight discomfort to the patient and is not desirable. This test was improved and shortened by using a system based on the oscillometric method of blood pressure measurement. By reducing the duration of the test from 5 minute reactive hyperemia to 3 minute reactive hyperemia, this will make the procedure practical for an increased number of patients, providing a noninvasive option to regularly check for early symptoms of cardiovascular disease.

Indiana University-Purdue University Indianapolis (IUPUI) / Study 1: Background: Insomnia may be a risk factor for cardiovascular disease in HIV (HIV-CVD); however, mechanisms have yet to be elucidated. Methods: We examined cross-sectional associations of insomnia symptoms with biological mechanisms of HIV-CVD (immune activation, systemic inflammation, and coagulation) among 1,542 people living with HIV from the Veterans Aging Cohort Study (VACS) Biomarker Cohort. Past-month insomnia symptoms were assessed by the item, “Difficulty falling or staying asleep?,” with the following response options: “I do not have this symptom” or “I have this symptom and…” “it doesn’t bother me,” “it bothers me a little,” “it bothers me,” “it bothers me a lot.” Circulating levels of the monocyte activation marker soluble CD14 (sCD14), inflammatory marker interleukin-6 (IL-6), and coagulation marker D-dimer were determined from blood specimens. Demographic- and fully-adjusted (CVD risk factors, potential confounders, HIV-related factors) regression models were constructed, with log-transformed biomarker variables as the outcomes. We present the exponentiated regression coefficient (exp[b]) and its 95% confidence interval (CI). Results: For sCD14 and D-dimer, we observed no significant associations. For IL-6, veterans in the “bothers a lot” group had 15% higher IL-6 than veterans in the “I do not have this symptom” group in the demographic-adjusted model (exp[b]=1.15, 95%CI=1.02-1.29, p=.03). This association was nonsignificant in the fully-adjusted model (exp[b]=1.07, 95%CI=0.95-1.19, p=.25). Conclusion: We observed little evidence of relationships between insomnia symptoms and markers of biological mechanisms of HIV-CVD. Other mechanisms may be responsible for the insomnia-CVD relationship in HIV; however, future studies with comprehensive assessments of insomnia symptoms are warranted. Study 2: Background: While insomnia has been identified as a potential risk factor for cardiovascular disease in HIV (HIV-CVD), research on the underlying pathophysiological mechanisms is scarce. Methods: We examined associations between 0-to-12-week changes in sleep disturbance and the concurrent 0-to-12-week changes and the subsequent 12-to-24-week changes in markers of systemic inflammation, coagulation, and endothelial dysfunction among people living with HIV (n = 33-38) enrolled in a depression clinical trial. Sleep disturbance was measured using the Pittsburgh Sleep Quality Index. Inflammatory markers interleukin-6 (IL-6) and C-reactive protein (CRP) and coagulation marker D-dimer were determined from blood specimens; endothelial dysfunction marker brachial flow-mediated dilation (FMD) was determined by ultrasound. 0-to-12-week variables were calculated as 12-week visit minus baseline, and 12-to-24-week variables were calculated as 24-week minus 12-week. We constructed multivariate linear regression models for each outcome adjusting for age, sex, race/ethnicity, Framingham risk score, and baseline depressive symptoms. Results: We did not observe statistically significant associations between 0-to-12-week changes in sleep disturbance and 0-to-12-week or 12-to-24-week changes in IL-6, CRP, D-dimer, or FMD. However, we did observe potentially meaningful associations, likely undetected due to low power. For 0-to-12-weeks, every 1-standard deviation (SD) increase, or worsening, in the sleep disturbance change score was associated with a 0.41 pg/mL and 80 ng/mL decease in IL-6 and D-dimer, respectively. For 12-to-24-weeks, every 1-SD increase in sleep disturbance change score was associated with a 0.63 mg/L, 111 ng/mL, and 0.82% increase in CRP, D-dimer, and FMD, respectively. Conclusion: We observed potentially meaningful, though not statistically significant, associations between changes in sleep disturbance and changes in biological mechanisms underlying HIV-CVD over time. Some associations were in the expected direction, but others were not. Additional studies are needed that utilize larger samples and validated, comprehensive assessments of insomnia.

With the high mortality rate of cardiovascular disease, it is important to study the early signs. The early detection of cardiovascular disease can lead to saved lives. Currently the most prevalent detection methods are the Framingham Risk Score and the carotid intima media thickness, both of which are insufficient. The necessary tool for early detection requires a uniform quantification system. The stimulus leading to endothelial dysfunction, the most significant predictor of a major adverse cardiovascular event (MACE)—and subsequently subclinical atherosclerosis—is reduced shear stress. Increased surface relative roughness affects the flow profile transition from laminar to turbulent resulting in reduced shear rate. The relationship between the shear stress and the relative roughness was studied using a computer model for fluid flow. A model of the brachial artery was generated to study its hemodynamics. Roughness values for both laminar and turbulent flow were calculated to use with the governing equations programmed in COMSOL Multiphysics. With all other factors remaining constant in the model, the roughness values were changed. From the model profile plots, line graphs, and numeral data are generated. This data provides information about how the shear stress and the shear rate change with respect to the relative roughness value. The models with different wall boundary conditions—slip versus Navier slip—were unable to be directly compared due to the differences in value magnitude. When the flow profile transitions from laminar to turbulent, there is a corresponding drop in both the shear stress and the shear rate values. Additional testing is required to determine a critical relative roughness value for this change in cumulative shear.

A surrogate arm model was created that is capable of being used for oscillometry. This model is capable of being used as a bench top model for blood pressure cuff devices. The arm consists of endplates and internal supports that are 3D printed with ABS, a silicone rubber outer sleeve, and interchangeable arteries made from two silicone rubber strips glued together at the edges. The interchangeable arteries have varying compliances that can be used as different inputs for oscillometric testing. A process was established to measure the artery compliances with a curve fit correlation of 0.95. However, testing revealed that this artery compliance relationship might not be an accurate representation of the artery compliance while it is in the surrogate arm system. A blood pressure cuff was also used with the surrogate arm model to measure changes in artery volume. Testing with the surrogate arm revealed a blood pressure cuff was capable of measuring artery volume changes of 2mL to 8mL consistently within 3.28% error. Volume changes of 1mL were unable to be repeatable measured accurately with a blood pressure cuff.

Yes / Endothelial dysfunction and damage underlie cerebrovascular disease and ischemic stroke. We undertook corneal confocal microscopy (CCM) to quantify corneal endothelial cell and nerve morphology in 146 patients with an acute ischemic stroke and 18 age-matched healthy control participants. Corneal endothelial cell density was lower (P<0.001) and endothelial cell area (P<0.001) and perimeter (P<0.001) were higher, whilst corneal nerve fbre density (P<0.001), corneal nerve branch density (P<0.001) and corneal nerve fbre length (P=0.001) were lower in patients with acute ischemic stroke compared to controls. Corneal endothelial cell density, cell area and cell perimeter correlated with corneal nerve fber density (P=0.033, P=0.014, P=0.011) and length (P=0.017, P=0.013, P=0.008), respectively. Multiple linear regression analysis showed a signifcant independent association between corneal endothelial cell density, area and perimeter with acute ischemic stroke and triglycerides. CCM is a rapid non-invasive ophthalmic imaging technique, which could be used to identify patients at risk of acute ischemic stroke. / Qatar National Research Fund Grant BMRP20038654

BACKGROUND: Type 2 diabetes (T2DM) is a metabolic disease that arises from insulin resistance and facilitates progression to cardiovascular consequences including myocardial infarction, coronary artery disease, and stroke. A contributor to the cardiovascular complications seen in T2DM is endothelial dysfunction. From a molecular standpoint, studies have shown that the pathophysiology of T2DM involves an altered metabolic milieu and increased oxidative stress, which both arise from insulin resistance, and lead to endothelial dysfunction. There is still much to discover on the pathways that are altered in this disease. Proteomics is a rapidly improving technique that can elucidate the differences in serum biomarkers, and their relationship to vascular endothelial health to further understand the pathophysiology of T2DM. OBJECTIVE: To evaluate the proteomic background and the implicated pathways in T2DM, and to understand how these biomarkers are associated with endothelial cell phenotype and systemic vascular function. METHODS: Age and sex similar individuals with T2DM and control individuals without T2DM between the ages of 30 and 80 were enrolled in this study. Blood was obtained for blood glucose and insulin levels and two proteomics panels assessing 192 serum biomarkers. Baseline vascular measures were obtained including blood pressure, heart rate, and flow-mediated dilation. Endothelial cells collected from participants were stimulated with insulin ex vivo and stained with phosphorylated endothelial nitric oxide synthase (p-eNOS) to measure changes in the insulin-mediated eNOS pathway. Associations between biomarker levels and insulin-stimulated p-eNOS levels were evaluated. RESULTS: The present study includes 69 subjects including 37 subjects with T2DM (age 57±8 years, 41% female) and 32 control subjects (age 53±9 years, 38% female). Measures of vascular health showed evidence of impairment in patients with T2DM including higher pulse pressure (56±12 mmHg versus 48±11 mmHg, p=0.02) and lower flow-mediated dilation (6.04±3.41% versus 9.1±4.4%, p=0.01). The proteomic panels revealed 24 serum biomarkers that were significantly upregulated and 2 that were significantly downregulated (adjusted p<0.05) in patients with T2DM compared to nondiabetic controls. These biomarkers are mainly involved in metabolism, vascular and fluid homeostasis, immune response, and apoptosis. Endothelial cell phenotype was abnormal in patients with T2DM compared to controls: mean fold change in insulin-stimulated p-eNOS was 0.34±0.07 for nondiabetic controls and -0.14±0.03 (p=0.01) for patients with T2DM. Renin and Adrenomedullin were significantly associated with lower insulin stimulated p-eNOS activation (r=-0.38, r=-0.27, and p=0.004, p=0.049 respectively). Whereas Chymotrypsin C (r=0.37, p=0.006), Paraoxonase 3 (r=0.35, p=0.009), Lipoprotein Lipase (r=0.34, p=0.01), and Superoxide Dismutase 2 (r=0.31, p=0.02) were significantly associated with higher insulin stimulated p-eNOS activation. CONCLUSIONS: We found associations between serum biomarker levels and insulin-stimulated p-eNOS levels which showed that there is a relationship between altered biomarkers and endothelial cell phenotype. Patients with T2DM had worse vascular endothelial health as shown by measures of endothelial dysfunction and arterial stiffness. Endothelial cell insulin resistance was present in patients with T2DM. In the same group, serum biomarkers showed elevated adiposity, inflammation and oxidative stress, and upregulation of the renin-angiotensin-aldosterone system.

yes / Type 2 diabetes (T2DM) confers increased risk of endothelial dysfunction, coronary heart disease, and vulnerability to vein graft failure after bypass grafting, despite glycaemic control. This study explored the concept that endothelial cells (EC) cultured from T2DM and nondiabetic (ND) patients are phenotypically and functionally distinct. Cultured human saphenous vein- (SV-) EC were compared between T2DM and ND patients in parallel. Proliferation, migration, and in vitro angiogenesis assays were performed; western blotting was used to quantify phosphorylation of Akt, ERK, and eNOS. The ability of diabetic stimuli (hyperglycaemia, TNF-α, and palmitate) to modulate angiogenic potential of ND-EC was also explored. T2DM-EC displayed reduced migration (~30%) and angiogenesis (~40%) compared with ND-EC and a modest, nonsignificant trend to reduced proliferation. Significant inhibition of Akt and eNOS, but not ERK phosphorylation, was observed in T2DM cells. Hyperglycaemia did not modify ND-EC function, but TNF-α and palmitate significantly reduced angiogenic capacity (by 27% and 43%, resp.), effects mimicked by Akt inhibition. Aberrancies of EC function may help to explain the increased risk of SV graft failure in T2DM patients. This study highlights the importance of other potentially contributing factors in addition to hyperglycaemia that may inflict injury and long-term dysfunction to the homeostatic capacity of the endothelium.

A Calibrated Cuff Plethysmography device was built, tested for verification, and used to experiment on human subjects to measure the cardiovascular response of consuming a beet supplement, specifically looking at arterial compliance and pressure-area curves. Each subject was tested four times. A baseline was measured under normal conditions and after five-minute hyperemia conditions. 10 subjects were given 6 ounces of water mixed with either purple Kool-Aid (control), a SuperBeets supplement, or a SuperBeets Sport supplement and after 45 minutes, measurements were taken undergoing normal and hyperemia conditions once more. The verification testing demonstrated the calibration of the device was effectively able to measure volume changes using a stationary metal pipe and IV bag, showing an average percent error of 3.11%. Data collected during the patient experiment resulted in the expected arterial compliance curves as well as pressure-area curves, when measurements were taken properly, and the subject didn’t move. These tests were able to validate the use of the device for measuring arterial compliance and seeing distinctions between normal and hyperemic conditions. However, many issues were presented and are thoroughly addressed in this paper for future research using the same device.

Cardiovascular disease is the leading cause of death globally and is responsible for taking 17.9 million lives per year. Despite the use of clinical treatments and detection methods, there remains a large population of individuals that suffer from CVD whose symptoms are left undetected and untreated prior to a life-threatening cardiac event. This highlights a need for an early detection method that can prevent the manifestation and worsening of the disease as well as address limitations of current early detection methods. An area of interest for early detection of CVD is subclinical atherosclerosis, which is the long, early, asymptomatic stage of plaque formation. Subclinical atherosclerosis has been namely associated with endothelial dysfunction and is the result of the pathological state of the endothelium due to its impact on vascular homeostasis, thrombosis, and vascular tone. Endothelial dysfunction is a result of several factors contributing to and promoting inflammation and results in changes in biological pathways that can alter the surface of the endothelium. This surface modification or added roughness changes the flow profile from laminar to turbulent flow due to the decreased shear stress on the vascular wall. Current detection methods such as carotid intima media thickness (CIMT) and flow-mediated dilation (FMD) targeted at identifying the early stages of atherosclerosis present limitations such as identifying late-stage effects of plaque formation and subjective readings highlight the need for a different approach to early detection. This experimental study aims to present a possible method of detecting the morphological changes of the endothelium due to inflammation through acoustic analysis of flow. Three silicone artery phantom groups were created with different degrees of inner diameter surface roughness to explore the relationship between relative roughness and sound associated with fluid flow. The results of this study are power spectral density graphs (PSD) which show frequency peaks associated with each of the phantoms at a theoretical laminar and turbulent Reynolds number. The PSD graphs show that there is a difference in frequency response between a smooth and rough artery phantom at the same flow rate providing preliminary support that sound analysis of fluid flow could provide information regarding early-stage cardiovascular disease.

Food insecurity is a growing issue in the United States. Iron deficiency is the most common form of nutritional deficiency in patients with endothelial dysfunction and vascular-related diseases. This preliminary study lays the groundwork for the “Nutrient deficiency-on-a-chip” model. Endothelial cells are cultured on mechanically tunable, enzymatically cross-linked gelatin and treated with deferoxamine, an iron chelator, or angiotensin II were used to simulate a nutrient deficient and diseased environment, respectively. As oxidative stress and disturbed barrier function are the most prevailing mechanism of angiotensin II and iron deficiency induced endothelial dysfunction, to test our model we investigated the changes in reactive oxygen species production and VE-cadherin expression in engineered endothelium. Both angiotensin II and deferoxamine treated engineered endothelium showed an increase in oxidative stress and disturbed barrier function. This in vitro model can be a useful tool to better understand disease mechanisms associated with nutrient deficiency and identify novel therapeutics.