The objective of these experiments is to explore the effect of LIPUS on the ultraexpansion and osteogenic differentiation of harvested passage-4 HUCPV-SCs. HUCPV-SCs were divided into two groups: a treatment group that received LIPUS for 10 minutes for 1, 7, and 14 days and a control group that received a sham treatment utilizing both basic and osteogenic media. The results in basic media and osteogenic media demonstrated nonsignificant differences in cell count, ALP, DNA content, and CD90. Statistically significant expression of OSP and PCNA was observed on day 14 in LIPUS treated group. Nucleostemin expression in the LIPUS-treated group was insignificant on days 1 and 7. However, a selective increase in osteogenic markers was obtained on day 7 for ALP and OCN and on day 14 for OPN. Future experiments are required to explore the effects of different application times and/or techniques of LIPUS on the behaviour of HUCPV-SCs. / Medical Science

Cerebral small vessel disease (SVD) is associated with increased risks of stroke and dementia, however the mechanisms remain unclear. Low cerebral blood flow (CBF) has long been suggested and accepted, but clinical evidence is conflicting. On the other hand, growing evidence suggests that increased intracranial pulsatility due to vascular stiffening might be an alternative mechanism. Pulse-gated phase-contrast MRI is an imaging technique that allows measuring of CBF contemporaneously with pulsatility in multiple vessels and cerebrospinal fluid (CSF) spaces. The overall aim of this thesis was to provide an overview of existing clinical evidence on both hypotheses, to test the reproducibility of CBF and pulsatility measures in phase-contrast MRI, and to explore the relationship between CBF and intracranial pulsatility and SVD features in a group of patients with minor stroke and SVD changes on brain imaging. I first systematically reviewed and meta-analysed clinical studies that have assessed CBF or intracranial pulsatility in SVD patients. There were 38 studies (n=4006) on CBF and 27 (n=3356) on intracranial pulsatility. Most were cross-sectional, and longitudinal studies were scarce. There were large heterogeneities in patient characteristics and indices used particularly for measuring and calculating pulsatility. Methods to reduce bias such as blinding and the expertise of structural image readers were generally poorly reported, and many studies did not account for the impact of confounding factors (e.g. age, vascular risk factors and disease severity) on CBF or pulsatility. Evidence for falling CBF predating SVD was not supported by longitudinal studies; high pulsatility in one large artery such as internal carotid arteries (ICA) or middle cerebral arteries might be related to SVD, but studies that measured arteries, veins and CSF in the same patients were very limited and the reliability of some pulsatility measures, especially in CSF, needs to be tested. In order to test the reproducibility of the CBF and intracranial pulsatility measures, I repeated 2D phase-contrast MRI scans of vessels and CSF on healthy volunteers during two visits. I also compared the ICA pulsatility index derived from the MRI flow waveform to that from the Doppler ultrasound velocity waveform in patients with minor stroke and SVD features. In 10 heathy volunteers (age 35.2±9.78 years), the reproducibility of CBF and vascular pulsatility indices was good, with within-subject coefficients of variability (CV) less than 10%; whereas CSF flow and pulsatility measures were generally less reproducible (CV > 20%). In 56 patients (age 67.8±8.27 years), the ICA pulsatility indices in Doppler ultrasound and MRI were acceptably well-correlated (r=0.5, p < 0.001) considering the differences in the two techniques. We carried out a cross-sectional study aiming to recruit 60 patients with minor stroke and SVD features. We measured CBF and intracranial pulsatility using phase-contrast MRI, as well as aortic augmentation index (AIx) using a SphygmoCor device. I first investigated the relationship between intracranial measures, and systemic blood pressure or aortic AIx, and then focused on how the intracranial haemodynamic measures related to two main SVD features (white matter hyperintensities (WMH) and perivascular spaces (PVS)). We obtained usable data from 56/60 patients (age 67.8±8.27 years), reflecting a range of SVD burdens. After the adjustment for age, gender, and history of hypertension, higher pulsatility in the venous sinuses was associated with lower diastolic blood pressure and lower mean arterial pressure (e.g. diastolic blood pressure on straight sinus pulsatility index (PI): β=-0.005, P=0.029), but not with aortic AIx. Higher aortic AIx was associated with low ICA PI (β=-0.011, P=0.040). Increased pulsatility in the venous sinuses, not low CBF, was associated with greater WMH volume (e.g. superior sagittal sinus PI: β=1.29, P=0.005) and more basal ganglia PVS (e.g. odds ratio=1.379 per 0.1 increase in superior sagittal sinus PI) after the adjustment for age, gender and blood pressure. The thesis is the first to summarise the literature on CBF and intracranial pulsatility in SVD patients, addressed the major limitations of current clinical studies of SVD, and also assessed CBF and intracranial pulsatility contemporaneously in well-characterised patients with SVD features. The overall results of the thesis challenge the traditional hypothesis of the cause and effect between low CBF and SVD, and suggest that increased cerebrovascular pulsatility, which might be due to intrinsic cerebral small vessel pathologies rather than just aortic stiffness, is important for SVD. More importantly, this pilot study also provides a reliable methodology for measuring intracranial pulsatility using phase-contrast MRI for future longitudinal or larger multicentre studies, and shows that intracranial pulsatility could be used as a secondary outcome in clinical trials of SVD. However, future research is required to elucidate the implication of venous pulsatility and to fully explore the passage of pulse wave transmission in the brain. Overall, this thesis advances knowledge and suggest potential targets for future SVD studies in terms of mechanisms, prevention and treatment.

No description available.

No description available.

Perivascular adipose (PVAT) has been hypothesized to influence arterial health, where an excess can lead to pathogenesis of atherosclerosis and other arterial pathologies. A novel assessment of carotid PVAT is the use of carotid extra media thickness (EMT) ultrasonography. Currently, there is a lack of research to demonstrate the relationship between carotid EMT and existing measures of arterial health, notably, central pulse wave velocity, and carotid distensibility and intimal media thickness. In the current cross sectional study, 81 participants of younger recreationally active (ages 23.2 ± 2.5 years), younger sedentary (ages 26.4 ± 7.2 years), older healthy (ages 70.3 ± 5.4 years) and older adults with coronary artery disease (CAD) (ages 67.9 ± 8.7 years) were recruited. Resting measures of central arterial stiffness was examined through the assessment of aPWV, while measures of local carotid stiffness were examined through carotid distensibility. Aortic PWV was calculated using an accepted direct distance method (80% of carotid to femoral direct distance) and time difference between the feet of the carotid and femoral waveforms. Carotid intima-media thickness (IMT), a measure of the inner arterial walls, and carotid extra media thickness (EMT), a measure of carotid PVAT, were assessed through B-mode ultrasound images and a semi-automated edge tracking software. Carotid EMT, IMT, and aPWV were significantly greater in older adults than in younger adults (p < 0.05). No difference in carotid EMT was found between younger recreationally active (0.47 ± .08 mm) and sedentary adults (0.46 ± .06 mm). There were also no differences in carotid EMT between the older healthy (0.58 ± .06 mm) and older adults with CAD (0.54 ± 0.08 mm). Carotid EMT was also significantly correlated with age (r =0 .500), waist circumference (r = 0.521), aPWV (r =0.431), carotid distensibility (r = -0.364 and IMT (r = 0.404). Despite significant correlations, carotid EMT was not an independent predictor of aPWV, carotid distensibility and IMT. Because of the lack of predictive power in measures of arterial stiffness and carotid IMT, there is a potential that carotid EMT may be an independent vascular disease marker. Future investigations should involve carotid EMT in longitudinal studies to evaluate the potential marker for a more comprehensive cardiovascular risk assessment. / Thesis / Master of Science (MSc)

No description available.

No description available.

Heart failure contributes to nearly 60,000 deaths per year in the USA and is often caused by hypertension and preceded by the development of left ventricular hypertrophy (LVH). LVH is usually accompanied by intensive interstitial and perivascular fibrosis which may contribute to arrhythmogenic sudden cardiac death. Emerging evidence indicates that LV dysfunction in patients and animal models of cardiac hypertrophy is closely associated with perivascular inflammation. To investigate the role of perivascular inflammation in coronary artery remodeling and cardiac fibrosis during hypertrophic ventricular remodeling, we used a well-established mouse model of pressure-overload-induced LVH: transverse aortic constriction (TAC). Early perivascular inflammation was indicated by accumulation of macrophages and T lymphocytes 24 hours post-TAC and which peaked at day 7. Coronary luminal platelet deposition was observed along with macrophages and lymphocytes at day 3. Also, LV protein levels of VEGF and MCP-1 were significantly increased. Consistent with lymphocyte accumulation, cardiac expression of IL-10 mRNA was elevated. Furthermore, circulating platelet-leukocyte aggregates tended to be higher after TAC, compared to sham controls. Platelets have been shown to modulate perivascular inflammation and may facilitate leukocyte recruitment at sites of inflamed endothelium. Therefore, we investigated the impact of thrombocytopenia in the response to TAC. Immunodepletion of platelets decreased early perivascular accumulation of T lymphocytes and IL-10 mRNA expression, and altered subsequent coronary artery remodeling. The contribution of lymphocytes was examined in Rag1-/- mice, which displayed significantly more intimal hyperplasia and perivascular fibrosis compared to wild-type mice following TAC. Collectively, our studies support a role of early perivascular accumulation of platelets and T lymphocytes in pressure overload-induced inflammation which will contribute to long-term LV remodeling. One potential mechanism for inflammatory cells to modulate their environment and affect surrounding cells is through release of cargo stored in granules. To determine the contribution of granule release from inflammatory cells in the development of LVH, we used Unc13dJinx (Jinx) mice, which contain a single point mutation in Unc13d gene resulting in defects in Munc13-4. Munc13-4 is a limiting factor in vesicular priming and fusion during granule secretion. Therefore, Jinx mice have defects in degranulation of platelets, NK cells, cytotoxic T lymphocytes, neutrophils, mast and other cells. With the use of bone marrow transplantation, Jinx chimeric mice were created to determine whether the ability of hematopoietic cells to secrete granule contents affects the development of LVH. Wild-type mice (WT) that were transplanted with WT bone marrow (WT>WT) and WT mice that received Jinx bone marrow (Jinx>WT) developed LVH and a classic fetal reprogramming response early after TAC (7 days), but at later times (5 weeks), Jinx>WT mice failed to sustain the cardiac hypertrophic response observed in WT>WT mice. No difference in cardiac fibrosis was observed at early or late times. Repetitive injection of WT platelets or platelet releasate restored cardiac hypertrophy in Jinx>WT mice. These results suggest that sustained LVH in the setting of pressure overload depends on factor(s) secreted, likely from platelets. In conclusion, our studies demonstrate that platelets and lymphocytes are involved in early perivascular inflammation post-TAC, which may contribute to later remodeling in the setting of LVH. Factors released from hematopoietic cells, including platelets, in a Munc13-4-dependent manner are required to promote cardiac hypertrophy. These findings focus attention on modulating perivascular inflammation and targeting granule cargo release to prevent the development and consequences of LVH.

Protein malnutrition (PM) causes anemia and leukopenia by reduction of hematopoietic precursors and impaired production of mediators that induce hematopoiesis, as well as structural and ultrastructural changes in the bone marrow (BM) extracellular matrix. Hematopoiesis occurs in the bone marrow (BM) in distinct regions called niches, which modulate the processes of differentiation, proliferation and self-renewal of the hematopoietic stem cell (HSC). The perivascular niche, composed mainly by mesenchymal stem cells (MSC) and endothelial cells (EC), is the major modulator of HSC and its function extends to the migration of mature hematopoietic cells into the peripheral blood through the production of cytokines and growth factors. Thus, our hypothesis is that PM changes the perivascular niche and our objective is to evaluate whether PM affects the modulatory capacity of MSC and EC on hematopoiesis. C57BL/6 male mice were divided into Control and Malnourished groups, which received for 5 weeks, respectively, a normal protein diet (12% casein) and a low protein diet (2% casein). After this period, animals were euthanized, nutritional and hematological evaluations were performed, featuring the PM. We performed leukemic myelo-monoblasts cells transplantation and observed that these cells have a lower proliferation rate and are rather in the cell cycle G0/G1 phases in malnourished mice, indicating that the BM microenvironment is compromised in PM. MSC were isolated, characterized and differentiated in vitro into EC cells, which were evidenced by CD31 and CD144 markers. We performed the quantification of HSC and hematopoietic progenitors, as well as some regulators of proliferation and differentiation, ex vivo and after cultures with MSC or EC. We observed that PM reduces HSC and hematopoietic progenitors ex vivo. In PM, MSC promote increase in HSC and suppress hematopoietic differentiation, whereas ECs induce cell cycle arrest. Additionally, we verified that PM affects granulopoesis by decreasing the expression of G-CSFr in granule-monocytic progenitors. Thus, we conclude that PD compromises hematopoiesis due to intrinsic alterations in HSC, as well as alterations in the medullary perivascular niche. / A desnutrição proteica (DP) provoca anemia e leucopenia decorrente da redução de precursores hematopoéticos e comprometimento da produção de mediadores indutores da hematopoese. A hematopoese ocorre na medula óssea (MO) em regiões distintas chamadas de nichos, que modulam os processos de diferenciação, proliferação e auto renovação da célula tronco hematopoiética (CTH). O microambiente perivascular, composto principalmente por células tronco mesenquimais (CTM) e células endoteliais (CE), é o principal modulador das CTH e sua função se estende até a migração das células hematopoiéticas maduras para o sangue periférico, através da produção de citocinas e fatores de crescimento. Dessa forma, nossa hipótese é que a DP altera o microambiente perivascular e objetivamos avaliar se a DP afeta a capacidade modulatória das CTM e CE sobre a hematopoese. Utilizamos camundongos C57BL/6 machos, divididos em grupos Controle e Desnutrido, sendo que o grupo Controle recebeu ração normoproteica (12% caseína) e o grupo Desnutrido recebeu ração hipoproteica (2% caseína), ambos durante 5 semanas. Após este período, os animais foram eutanasiados, foi realizada a avaliação nutricional e hematológica, caracterizando a DP. Realizamos transplantes de mielomonoblastos leucêmicos e observamos que estas células apresentam menor taxa de proliferação e se encontram em maior quantidade nas fases G0/G1 do ciclo celular em camundongos desnutridos, indicando que o microambiente medular está comprometido. Isolamos CTM, que foram caracterizadas e diferenciadas in vitro em CE, o que foi evidenciado pelos marcadores CD31 e CD144. Quantificamos CTH e progenitores hematopoéticos, bem como reguladores de proliferação e diferenciação, ex vivo e após culturas com CTM ou CE. Observamos que a DP reduz CTH e progenitores hematopoéticos ex vivo. Na DP, as CTM promovem incremento de CTH e suprimem a diferenciação hematopoética, enquanto que as CE induzem parada no ciclo celular. Adicionalmente, observamos que a DP afeta a granulopoese por diminuição da expressão de G-CSFr nos progenitores grânulo-monocíticos. Dessa forma, concluímos que a DP compromete a hematopoese por alterações intrínsecas na CTH, como também por alterações ocasionadas no microambiente perivascular medular.

Heart failure (HF) induced by aortic pressure over-load is associated with increased coronary artery stiffness. Perivascular adipose tissue (PVAT) and advanced glycation end products (AGE) both promote arterial stiffness. However, the mechanisms by which coronary PVAT promotes arterial stiffness and the efficacy of exercise to prevent coronary stiffness are unknown. The present study hypothesized both chronic continuous and interval exercise training would prevent coronary artery stiffness associated with inhibition of PVAT secreted AGE. Yucatan mininature swine were divided into four groups: control-sedentary (CON), aortic-banded sedentary heart failure (HF), aortic-banded HF continuous exercise trained (HF+CONT), and aortic-banded HF interval exercise trained (HF+IT). Coronary artery stiffness was assessed by ex vivo mechanical testing and coronary artery elastin, collagen and AGE-related proteins were assessed by immunohistochemistry. HF promoted coronary artery stiffness with reduced elastin content and greater AGE accumulation which was prevented by chronic continuous and interval exercise training. HF PVAT secreted higher AGE compared with CON and was prevented in the HF+CONT and HF+IT groups. Young healthy mouse aortas cultured in HF PVAT conditioned media had increased stiffness, lower elastin content and AGE accumulation compared with CON, which was prevented by PVAT from the HF+CONT and HF+IT groups. HF coronary PVAT secreted greater interleukin-6 (IL-6) and IL-8 compared to CON which was prevented by both continuous and interval exercise training regimens. We conclude chronic continuous and interval exercise is a potential therapeutic strategy to prevent coronary artery stiffness via inhibition of PVAT-derived AGE secretion in a pre-clinical mini-swine model of pressure overload-induced HF.