To this day heart failure with preserved ejection fraction (HFpEF) remains a poorly understood malady. Half of all heart failure (HF) cases are HFpEF, and the prevalence of HF is on the rise. Unlike HF with reduced ejection fraction, HFpEF has no treatment options and is often times difficult to diagnose because victims of HFpEF often have pre-existing conditions. Vascular endothelial growth factor (VEGF) has been implicated in maintaining myocardial health and is thought to play a role in HFpEF. We sought to test the hypothesis that VEGF-A plays a role in HFpEF in a hypertensive murine model of HFpEF. Using Western blot analysis we found that there was an up regulation of VEGF-A in the homogenized left ventricle (LV) of our HFpEF mice. Unexpectedly, there was a down regulation of VEGF-A in the homogenized tissue from the aorta in those mice. To study the circulating levels of VEGF in our HFpEF mice we used an ELISA. We found that our HFpEF mice had similar levels of circulating VEGF as our control. This suggests that VEGF has paracrine/autocrine role in our HFpEF model rather than endocrine, like our human data suggested. To identify the cells responsible for the expression profile we saw in the homogenized tissue data we looked at the response of adult rat ventricular myocytes (ARVM) and vascular smooth muscle cells (VSMC) to aldosterone stimulation at short (1hr) and long (24hr) time points at both physiological (50nm) and pathological (1μm) concentrations. To do this analysis we recruited the help of Western blot, ELISA and RT-PCR techniques to construct a consistent VEGF expression profile. The Western blot ARVM data showed statistically significant (P<0.05) increase in VEGF-A to pathological doses of aldosterone, especially at the longer time point. When we tested the VSMC using Western blot analysis, we found that the trend of our n=1 sample suggested a strong response to the physiological dose of aldosterone in the short term. Using the more sensitive ELISA technique to measure the VEGF content of our VCMS we increasing our sample size to n=4 and found no statistically significant (p=NS) response to aldosterone stimulation from the VSMC. However, looking at the trends in the data it is clear that VSMC increases VEGF in response to long-term physiological doses of aldosterone. This is contrary to what we found using Western blot analysis, so we queried the VEGF mRNA from the VSMC to settle the score. Unfortunately, this too proved fruitless. The RT-PCR data was not significant and the trend was that of the ARVM expression profile. We initially turned to VSMC because we hypothesized that they could contribute to the paracrine/autocrine activity similar to what we saw in the LV from the ARVM. It is unclear if VSMC play a role in HFpEF progression, but their lack of consistent response to aldosterone could potential explain the down regulation of VEGF-A we observed in the aorta of our HFpEF mice. We initially sough to test the hypothesis that VEGF-A plays a role in our HFpEF mouse model, what we found was that ARVM contribute to localized VEGF-A increased production in the LV while in the aorta there is a down regulation of VEGF-A in our HFpEF model, we are unable to make any conclusion about VSMC response to aldosterone because of insufficient sample size. Thus in conclusion, it appears that VEGF-A does play a role in our HFpEF model specifically in a paracrine/autocrine manner in the LV where the ARVM contributes to the increased production of the cytokine.

<p>Consequences of atherosclerosis may result in a number of diseases of the cardiovascular system that represent serious health problems and major causes of morbidity and mortality worldwide. Although it is initially considered as disease of fibro-lipid and thrombus deposition in the arterial wall, it also involves an ongoing inflammatory response.</p><p>Normally, the inflammatory response is considered as a protective defence mechanism of the body. However, if the inflammation gets out of proportion to the threat it is dealing with, it may then result in a sustained chronic disorder and thus may underlie the initial stage of atherogenesis. The work of this thesis focuses on the expression of cytokines/chemokines and the vascular transcriptional response to inflammation, i.e. LPS in atherosclerosis. This has mainly been studied in animal models of atherosclerosis; consequently, we set out to investigate these events using human material in vitro (human carotid lesions).</p><p>Employing quantitative analysis, we were able to detect a significant induction of protein and mRNA of the cytokines IL-1β, IL-6, IL-10 and TNF-α and the chemokines IL-8 and MCP-1 by LPS in both atherosclerotic and non-atherosclerotic vessels. In contrast, LPS induction of TNF-α, IL-1β and IL-10 was solely observed in the lesions, but not in normal arteries. In addition, the impact of IL-1 gene polymorphism on the risk of myocardial infarction (MI) was estimated by DNA genotyping of 387 survivors of a first MI and 387 sex and age-matched control subjects. We found no statistically significant differences in either genotypic distribution or allelic frequencies of IL-1β (-511) or IL-1Ra (VNTR) polymorphisms between first-time survivors of myocardial infarction and their age-matched healthy controls. Incontrast, our results demonstrated a strong association between the IL-1Ra genotype and severity of angiographically determined coronary artery disease in post-MI patients. To further investigate the vascular response to inflammation, we used gene array analysis to evaluate the human vascular transcriptional response to LPS of non-atherosclerotic human renal arteries compared to carotid lesions. In LPS treated renal arteries, 54% of the transcripts gave a detectable signal, where 4% were upregulated and 3.8% down-regulated. In the LPS stimulated carotid lesions, 44% of transcripts were detected. In this latter group, 5.1% of transcripts were increased and 3.3% decreased. Interestingly, a newly identified virus-inducible antiviral protein, CMV inducible gene ₅/viperin (Cig₅), was among the most strongly induced gene in both normal and atherosclerotic biopsies. Single gene analysis revealed viperin in the endothelium of human atherosclerotic lesions. Further, viperin was induced in vascular cells by inflammatory stimuli and CMV infection.</p><p>In conclusion we show that atherosclerotic vessels produce more proinflammatory cytokines/chemokines than normal vessels. Interestingly, our results indicate that LPS enhances the expression of cytokines/chemokines in a similar pattern both in lesions and normal arteries. However, the response is stronger in atherosclerotic lesions. Furthermore, our results suggest that genetic polymorphisms within the IL-1Ra loci may influence the severity of CAD. Finally, the CMV inducible gene ₅/viperin have been identified as a putative culprit molecule in vascular inflammation and atherosclerosis.</p>

<p>Methylglyoxal (MG) is a reactive glucose metabolite and a known causative factor for hypertension and diabetes. Hydrogen sulfide (H₂S), on the other hand, is a gasotransmitter with multifaceted physiological functions, including anti-oxidant and vasodilatory properties. The present study demonstrates that MG and H₂S can interact with and modulate each other's functions. Upon <i>in vitro</i> incubations, we found that MG and H₂S can directly interact to form three possible MG-H₂S adducts. Furthermore, the endogenous production level of MG or H₂S was significantly reduced in a concentration-dependent manner in rat vascular smooth muscle cells (A-10 cells) treated with NaHS, a H₂S donor, or MG, respectively. Indeed, MG-treated A-10 cells exhibited a concentration-dependent down-regulation of the protein and activity level of cystathionine &gamma;-lyase (CSE), the main H₂S-generating enzyme in the vasculature. Moreover, H₂S can induce the inhibition of MG-generated ROS production in a concentration-dependent manner in A-10 cells. In 6-22 week-old CSE knockout male mice (CSE^-/-), mice with lower levels of vascular H₂S, we observed a significant elevation in MG levels in both plasma and renal extracts. Renal triosephosphates were also significantly increased in the 6-22 week-old CSE^-/- mice. To identify the source of the elevated renal MG levels, we found that the activity of fructose-1,6-bisphosphatase (FBPase), the rate-limiting enzyme in gluconeogenesis, was significantly down-regulated, along with lower levels of its product (fructose-6-phosphate) and higher levels of its substrate (fructose-1,6-bisphosphate) in the kidney of 6-22 week-old CSE^-/- mice. We have also observed lower levels of the gluconeogenic regulator, peroxisome proliferator-activated receptor-&gamma; coactivator (PGC)-1&alpha;, and its down-stream targets, FBPase-1 and -2, phosphoenolpyruvate carboxykinase (PEPCK), and estrogen-related receptor (ERR)&alpha; mRNA expression levels in renal extracts from 6-22 week-old CSE^-/- mice. Likewise, FBPase-1 and -2 mRNA levels were also significantly down-regulated in aorta tissues from 14-16 week-old CSE^-/- mice. Administration of 30 and 50 &#x00B5;M NaHS induced a significant increase in FBPase-1 and PGC-1&alpha; in rat A-10 cells. We have also observed a significant up-regulation of PEPCK and ERR&alpha; mRNA expression levels in 50 &#x00B5;M NaHS-treated A-10 cells, further confirming the involvement of H₂S in regulating the rate of gluconeogenesis and MG formation. Overall, this unique study demonstrates the existence of a negative correlation between MG and H₂S in the vasculature. Further elucidation of this cross-talk phenomenon between MG and H₂S could lead to more elaborate and effective therapeutic regimens to combat metabolic syndrome and its related health complications.</p>

<p>Methylglyoxal (MG) is a reactive glucose metabolite and a known causative factor for hypertension and diabetes. Hydrogen sulfide (H₂S), on the other hand, is a gasotransmitter with multifaceted physiological functions, including anti-oxidant and vasodilatory properties. The present study demonstrates that MG and H₂S can interact with and modulate each other's functions. Upon <i>in vitro</i> incubations, we found that MG and H₂S can directly interact to form three possible MG-H₂S adducts. Furthermore, the endogenous production level of MG or H₂S was significantly reduced in a concentration-dependent manner in rat vascular smooth muscle cells (A-10 cells) treated with NaHS, a H₂S donor, or MG, respectively. Indeed, MG-treated A-10 cells exhibited a concentration-dependent down-regulation of the protein and activity level of cystathionine &gamma;-lyase (CSE), the main H₂S-generating enzyme in the vasculature. Moreover, H₂S can induce the inhibition of MG-generated ROS production in a concentration-dependent manner in A-10 cells. In 6-22 week-old CSE knockout male mice (CSE^-/-), mice with lower levels of vascular H₂S, we observed a significant elevation in MG levels in both plasma and renal extracts. Renal triosephosphates were also significantly increased in the 6-22 week-old CSE^-/- mice. To identify the source of the elevated renal MG levels, we found that the activity of fructose-1,6-bisphosphatase (FBPase), the rate-limiting enzyme in gluconeogenesis, was significantly down-regulated, along with lower levels of its product (fructose-6-phosphate) and higher levels of its substrate (fructose-1,6-bisphosphate) in the kidney of 6-22 week-old CSE^-/- mice. We have also observed lower levels of the gluconeogenic regulator, peroxisome proliferator-activated receptor-&gamma; coactivator (PGC)-1&alpha;, and its down-stream targets, FBPase-1 and -2, phosphoenolpyruvate carboxykinase (PEPCK), and estrogen-related receptor (ERR)&alpha; mRNA expression levels in renal extracts from 6-22 week-old CSE^-/- mice. Likewise, FBPase-1 and -2 mRNA levels were also significantly down-regulated in aorta tissues from 14-16 week-old CSE^-/- mice. Administration of 30 and 50 &#x00B5;M NaHS induced a significant increase in FBPase-1 and PGC-1&alpha; in rat A-10 cells. We have also observed a significant up-regulation of PEPCK and ERR&alpha; mRNA expression levels in 50 &#x00B5;M NaHS-treated A-10 cells, further confirming the involvement of H₂S in regulating the rate of gluconeogenesis and MG formation. Overall, this unique study demonstrates the existence of a negative correlation between MG and H₂S in the vasculature. Further elucidation of this cross-talk phenomenon between MG and H₂S could lead to more elaborate and effective therapeutic regimens to combat metabolic syndrome and its related health complications.</p>

Coronary artery stent placement has been a significant advance in the percutaneous treatment of atherosclerotic disease, and tissue engineered vascular grafts may provide a viable alternative to autologous segments for small diameter vessels. However, in-stent restenosis remains an important limitation, and tissue engineered grafts have poor patency and high risk of thrombus formation due to their inability to maintain a confluent, adherent, and quiescent endothelium. While animal models provide insight into the pathophysiology of these situations, elucidation of the relative importance of stent or graft components, hemodynamic factors, and molecular factors is difficult. Very little research has focused on bridging gaps in knowledge concerning blood/biomaterial interactions, blood/endothelial cell interactions, and endothelial cell/smooth muscle cell cross-talk. The work presented within this thesis will do just that. The objective of this thesis research was to elucidate the influence of biomaterial-induced activation of leukocytes on endothelial cell or smooth muscle cell phenotype, as well as endothelial cell/smooth muscle cell cross-talk in co-culture systems. Towards this goal, two complimentary in vitro endothelial cell/smooth muscle cell co-culture models with divergent smooth muscle cell phenotype were developed and characterized. Using these systems, it was found that the presence of more secretory smooth muscle cells (as would be seen in wound healing or disease) in general enhanced endothelial cell activation in response to biomaterial-pretreated monocytes, while the presence of less secretory smooth muscle cells (to model more quiescent smooth muscle cells found in uninjured healthy vessels) suppressed endothelial cell activation in response to biomaterial-pretreated monocytes (and neutrophils to a small degree). Additionally, biomaterial-pretreated monocytes and neutrophils amplified a smooth muscle cell phenotypic shift away from a more quiescent state. It is likely that the compounding effect of secretory smooth muscle cells and biomaterial-activated leukocytes are responsible for altered vascular wound healing upon implantation of stents or vascular grafts. Understanding the specific signals causing these effects, or signals delivered by contractile smooth muscle cells that limit these effects help to provide design criteria for development of devices or grafts capable of long term patency.

One of the major causes of death in developed countries is cardiovascular disease that affects small and medium sized blood vessels. In most cases autologous grafts have to be used which have limited availability. A functional tissue engineered vessel can be the ultimate solution for vascular reconstruction. Tissue engineered constructs with cells growing in an organized manner have been shown to have improved mechanical properties. In the present study collagen scaffolds with 650 nm, 500 nm and 332.5 nm wide channels and ridges were seeded with human vascular smooth muscle cells (VSMC) and human endothelial cells seperately and then co-cultured on tubular scaffolds. When the films were seeded with endothelial cells it was observed that nanopatterns do not affect cell proliferation or initial cell alignment / however, they significantly influenced cell retention under shear (fluid flow). While 35 &plusmn / 10 % of the cells were retained on unpatterned films, 75 &plusmn / 4 % was retained on 332.5 nm patterned films and even higher, 91 &plusmn / 5 % was retained on 650 nm patterned films. It was shown that nanopatterns as small as 332.5 nm could align the vascular smooth muscle cells (VSMC) and that alignment significantly improved mechanical properties. Presence of nanopatterns increased the ultimate tensile strength (UTS) from 0.55 &plusmn / 0.11 on Day 0 to as much as 1.63 &plusmn / 0.46 MPa on Day 75, a value within the range of natural arteries and veins. Similarly, Young&amp / #8217 / s Modulus values were ca. 4 MPa, again in the range of the natural vessels. Since the films would be ultimately rolled into tubes of collagen, nutrient transfer through the films is quite crucial. Diffusion coefficient for 4-acetaminophenol and oxygen through the collagen films were found to be 1.86 &plusmn / 0.39 x 10-7 cm2.s-1 and 5.41 &plusmn / 2.14 x 10-7 cm2.s-1, repectively in the unseeded form, and increased by 4 fold after cell seeding, which is comparable to that in natural tissues. When both cell types were co-cultured on the nanopatterned tubes (a both-side nanopatterned collagen tube), it was shown that on the outside of the tube VSMCs proliferated in an oriented manner and on the inside endothelial cells proliferated as a monolayer. Therefore, this study showed that cell guidance enhances the mechanical properties of engineered vessels, and help overcome the two most important challenges in vascular tissue engineering / the need for adequate mechanical properties and continuous lining of endothelial cells even under physiological shear stress.

AIMS: Macrophages (MPs) and vascular smooth muscle cells (VSMCs) closely interact within the growing atherosclerotic plaque. An in vitro co-culture model was established to study how MPs modulate VSMC behaviour. METHODS AND RESULTS: MPs were exposed to fluorescence-labelled-acetylated LDL (FL-acLDL) prior to co-culture with VSMCs. Fluorescence microscopy visualized first transport of FL-acLDL within 6 h after co-culture implementation. When MPs had been fed with FL-acLDL in complex with fluorescence-labelled cholesterol (FL-Chol), these complexes were also transferred during co-culture and resulted in cholesterol positive lipid droplet formation in VSMCs. When infected with a virus coding for a fusion protein of Rab5a and fluorescent protein reporter (FP) to mark early endosomes, no co-localization between Rab5a-FP and the transported FL-acLDL within VSMCs was detected implying a mechanism independent of phagocytosis. Next, expression of lysosome-associated membrane glycoprotein 1 (LAMP1)-FP, marking all lysosomes in VSMCs, revealed that the FL-acLDL was located in non-acidic lysosomes. MPs infected with virus encoding for LAMP1-FP prior to co-culture demonstrated that intact fluorescence-marked lysosomes were transported into the VSMC, instead. Xenogenic cell composition (rat VSMC, human MP) and subsequent quantitative RT-PCR with rat-specific primers rendered induction of genes typical for MPs and down-regulation of the cholesterol sensitive HMG-CoA reductase. CONCLUSION: Our results demonstrate that acLDL/cholesterol-loaded lysosomes are transported from MPs into VSMCs in vitro. Lysosomal transfer results in a phenotypic alteration of the VSMC towards a foam cell-like cell. This way VSMCs may lose their plaque stabilizing properties and rather contribute to plaque destabilization and rupture.

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Consequences of atherosclerosis may result in a number of diseases of the cardiovascular system that represent serious health problems and major causes of morbidity and mortality worldwide. Although it is initially considered as disease of fibro-lipid and thrombus deposition in the arterial wall, it also involves an ongoing inflammatory response. Normally, the inflammatory response is considered as a protective defence mechanism of the body. However, if the inflammation gets out of proportion to the threat it is dealing with, it may then result in a sustained chronic disorder and thus may underlie the initial stage of atherogenesis. The work of this thesis focuses on the expression of cytokines/chemokines and the vascular transcriptional response to inflammation, i.e. LPS in atherosclerosis. This has mainly been studied in animal models of atherosclerosis; consequently, we set out to investigate these events using human material in vitro (human carotid lesions). Employing quantitative analysis, we were able to detect a significant induction of protein and mRNA of the cytokines IL-1β, IL-6, IL-10 and TNF-α and the chemokines IL-8 and MCP-1 by LPS in both atherosclerotic and non-atherosclerotic vessels. In contrast, LPS induction of TNF-α, IL-1β and IL-10 was solely observed in the lesions, but not in normal arteries. In addition, the impact of IL-1 gene polymorphism on the risk of myocardial infarction (MI) was estimated by DNA genotyping of 387 survivors of a first MI and 387 sex and age-matched control subjects. We found no statistically significant differences in either genotypic distribution or allelic frequencies of IL-1β (-511) or IL-1Ra (VNTR) polymorphisms between first-time survivors of myocardial infarction and their age-matched healthy controls. Incontrast, our results demonstrated a strong association between the IL-1Ra genotype and severity of angiographically determined coronary artery disease in post-MI patients. To further investigate the vascular response to inflammation, we used gene array analysis to evaluate the human vascular transcriptional response to LPS of non-atherosclerotic human renal arteries compared to carotid lesions. In LPS treated renal arteries, 54% of the transcripts gave a detectable signal, where 4% were upregulated and 3.8% down-regulated. In the LPS stimulated carotid lesions, 44% of transcripts were detected. In this latter group, 5.1% of transcripts were increased and 3.3% decreased. Interestingly, a newly identified virus-inducible antiviral protein, CMV inducible gene 5/viperin (Cig5), was among the most strongly induced gene in both normal and atherosclerotic biopsies. Single gene analysis revealed viperin in the endothelium of human atherosclerotic lesions. Further, viperin was induced in vascular cells by inflammatory stimuli and CMV infection. In conclusion we show that atherosclerotic vessels produce more proinflammatory cytokines/chemokines than normal vessels. Interestingly, our results indicate that LPS enhances the expression of cytokines/chemokines in a similar pattern both in lesions and normal arteries. However, the response is stronger in atherosclerotic lesions. Furthermore, our results suggest that genetic polymorphisms within the IL-1Ra loci may influence the severity of CAD. Finally, the CMV inducible gene 5/viperin have been identified as a putative culprit molecule in vascular inflammation and atherosclerosis.

The underlying inflammatory storm in renal or diabetic disease may induce expression of inducible nitric oxide synthase (iNOS). Similarly, expression of iNOS or nitric oxide (NO) production in vascular smooth muscle cells (VSMCs) in a calcifying environment, may promote vascular calcification (VC) (Zaragoza et al., 2006). However, emerging data suggests that NO generated by either endothelial nitric oxide synthase (eNOS) or iNOS may protect VSMCs from VC (Kanno et al., 2008). Thus, the role of NO and its associated enzymes in the development of VC is unclear. The aim of this study was to identify whether NO produced by iNOS regulates calcification in VSMCs, and to further understanding of potential mechanisms that may mediate the actions of NO/iNOS. A significant and sustained production of NO by iNOS, which peaked at day 3 and declined thereafter was found in rat aortic smooth muscle cells (RASMCs) that were preactivated with lipopolysaccharide (LPS; 100μg ml-1) and interferon gamma (IFN-γ;100U ml-1) in the presence of calcification buffer (CB) containing calcium chloride (CaCl2; 7mM) and β-glycerophosphate (β-GP; 7mM). This was associated with formation of hydroxyapatite crystals (HA) or calcification plaques, observed via alizarin red staining (ARS) and/or fourier transform infrared (FT-IR) analysis. However, when RASMCs were incubated with the iNOS inhibitor GW274150 at 10 μM, together with LPS + IFN-γ + CB, HA crystal formation was abolished. When RASMCs were pretreated with diethylenetriamine/nitric oxide adduct (NOC 18) at either 30 or 50 μM for an hour prior to addition of CB, to generate NO; calcium levels were elevated leading to form HA crystals. However, the elevation of calcium caused by the presence of NO generated via iNOS, did not result in phosphorylation of mitogen activated protein kinases (p38 MAPK), extracellular signal-regulated kinases (Erks), and protein kinase B. Furthermore, there was a reduction of Runx2 levels (pro-calcific factor) which could be another pro-calcific factor involved in this mechanism. These findings suggest that NO may indeed play a fundamental role in calcification, enhancing mineralisation of smooth muscle cells. Furthermore, the expression of iNOS/ NO appears to be enhanced under conditions that favour calcification and these together may contribute to enhanced calcification with potential detrimental consequences in vivo.