Angiogenic cell therapy is currently being developed as a treatment for coronary artery disease (CAD); however, endothelial dysfunction (ED), commonly found in patients with CAD, impairs the ability for revascularization to occur. We hypothesized that culture on a collagen matrix will improve survival and function of circulating progenitor cells (CPCs) isolated from a mouse model of ED. Overall, ED decreased the expression of endothelial markers in CPCs and impaired their function, compared to normal mice. Culture of CPCs from ED mice on collagen was able to increase cell marker expression, and improve migration and adhesion potential, compared to CPCs on fibronectin. Nitric oxide production was reduced for CPCs on collagen for the ED group; however, CPCs on collagen had better viability under conditions of serum deprivation and hypoxia, compared to fibronectin. This study suggests that a collagen matrix may improve the function of therapeutic CPCs that have been exposed to ED.

A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine. / Primary pulmonary hypertension (PPH) has an extremely poor prognosis with a mean survival time of 2‐3 years from time of diagnosis. Hemodynamically, PPH is defined with a mPAP of ≥ 25 mm Hg. Currently, RHC is the gold standard for measuring the arterial pressures and diagnosing PPH; however, it is an incredibly invasive procedure. Our study will show whether CT angiography can be considered as a non‐invasive alternative for diagnosing PPH. Studies in the past have shown CT measurements of the MPAD and MPAD/AAD ratio having strong correlations with PPH. In addition to those measurements, we want to show if other CT parameters also have a correlation with PPH. Some of these novel measurements include the interventricular septal deviation and the Elizabeth Taylor sign. The interventricular septum is normally bowing to the right in a non‐pathological state. If it is straight or bowing to the left, this will indicate increased right ventricular pressures which would be indicative of PPH. Straight will indicate increased RV pressures, and bowing to the left will be considered markedly increased RV pressures. The Elizabeth Taylor sign is the ratio of the diameter of the segmental bronchi and its corresponding artery. We will hypothesize that the artery will be much larger than the bronchi in patients with PPH. Other measurements will include the left and right pulmonary arteries. This study is a retrospective review of subjects who underwent an otherwise unremarkable CT pulmonary artery angiogram. Subjects with pulmonary embolism or other acute pulmonary diseases are excluded. For each subject, the following CT findings are obtained: main pulmonary artery diameter (mPAD), ratio of mPAD to ascending aorta, right and left pulmonary artery diameters, ratio of segmental pulmonary artery to corresponding bronchus, and interventricular septal displacement. Straightening of the interventricular septum qualifies as increased right ventricular septal pressure and right‐to‐left bowing of the septum qualifies as a marked increase. Mean pulmonary artery pressure measured on any prior/subsequent RHC or echocardiogram within 3 months of the CT is recorded. Any past medical history of connective tissue disease is noted. Descriptive data are calculated and correlations are done to assess for presence and strength of associations among variables. Data from 484 subjects are collected. Incidence rate of pulmonary hypertension isv13% (n=63). 52% (n=33) of the subjects with pulmonary hypertension are female with an average age of 55 years. mPA diameter (p<0.001), mPA:AA ratio (p<0.001), right (p<0.001) and left pulmonary artery (p=0.004) diameters are predictors of pulmonary hypertension. sPA:B ratio (p=0.08) and interventricular septal displacement (p=0.96) are not predictive of pulmonary hypertension. This study supports an association of mPA diameter, mPA:AA ratio, right and left pulmonary artery diameters with pulmonary hypertension diagnosed by RHC or echocardiogram. Prospective research is warranted to confirm and establish threshold values for each variable. Currently, an invasive RHC remains the most accurate method of diagnosis. Correlating CT findings with pulmonary hypertension would allow clinicians to use CT as a noninvasive screening tool.

Hypoxia-induced pulmonary hypertension (HPH) is a progressive disease that is mainly caused by chronic exposure to high altitude, chronic obstructive lung disease, and obstructive sleep apnea. The increased pulmonary vascular resistance and increased pulmonary arterial pressure result in increased right ventricular afterload, leading to right heart failure and increased morbidity. There are several clinical reports suggesting a link between PH and diabetes, insulin resistance, or obesity; however, it is unclear whether HPH is associated with diabetes as a progressive complication in diabetes. The major goal of this study is to examine the effect of diabetic ''preconditioning'' or priming effect on the progression of HPH and define the molecular mechanisms that explain the link between diabetes and HPH. Our data show that HPH is significantly enhanced in diabetic mice, while endothelium-dependent relaxation in pulmonary arteries is significantly attenuated in chronically hypoxic diabetic mice (DH). In addition, we demonstrate that mouse pulmonary endothelial cells (MPECs) isolated from DH mice exhibit a significant increase in mitochondrial reactive oxygen species (ROS) concentration and decreased SOD2 protein expression. Finally, scavenging mitochondrial ROS by mitoTempol restores endothelium-dependent relaxation in pulmonary arteries that is attenuated in DH mice. These data suggest that excessive mitochondrial ROS production in diabetic MPECs leads to the development of severe HPH in diabetic mice exposed to hypoxia.

The endothelial nitric oxide synthase (eNOS) co-factor tetrahydrobiopterin (BH4) has been shown to play a pivotal role in maintaining endothelial function in experimental vascular disease models. In BH4-deficient states, eNOS becomes enzymatically ‘uncoupled’, generating reactive oxygen species instead of nitric oxide, thus promoting endothelial dysfunction. In humans with coronary artery disease (CAD), higher vascular BH4 levels have been shown to be associated with improved endothelial function, and genetic variation in endogenous BH4 synthesis has implicated a causal role. Accordingly, BH4 has been proposed as a potential therapeutic target in vascular disease states. The work in this thesis aims to further elucidate the roles of exogenous and endogenous BH4 in humans. In a randomised, placebo-controlled clinical trial of oral BH4 therapy in patients with CAD, exogenous BH4 had no effect on endothelial function or vascular oxidative stress. Subsequent pharmacokinetic and pharmacodynamic analysis revealed that oral BH4 significantly augmented BH4 levels in plasma and in venous tissue (but not in arterial tissue), but also increased levels of the oxidation product dihydrobiopterin (BH2), which lacks eNOS cofactor activity. Thus, there was a null effect on overall biopterin redox status. To further understand the mechanics of exogenous BH4 oxidation, ex vivo studies of human blood and vascular tissue demonstrated that exogenous BH4 is very rapidly oxidised to BH2; co-administration with an antioxidant had only a modest effect on preventing BH4 oxidation in blood, with no beneficial effect on biopterin redox state in the vasculature. Finally, using a “Mendelian randomisation” approach, I studied the effects of a haplotype of GCH1 (the gene encoding the rate limiting enzyme in BH4 synthesis) on endogenous BH4 bioavailability and vascular function in healthy individuals. In patients with CAD, this haplotype has been associated with decreased BH4 bioavailability and eNOS uncoupling, however in healthy individuals the haplotype exerted no significant effect, likely due to reduced inflammatory stimulation of GCH1.

No description available.

During dynamic exercise the arterial baroreflexes have been thought to reset to the prevailing level of systemic pressure in order to modulate transient changes in blood pressure with the same sensitivity (gain) as at rest. To test this hypothesis, cardiovascular responses to dynamic exercise and carotid baroreflex responses to graded neck suction and neck pressure (NS/NP) were examined in seven men of moderate fitness (V02 = 41.4±3.6 ml O2*kg^-1*min^-1) during two levels (20% and 40% of peak oxygen uptake) of steady-state exercise. In addition, deactivation of cardiopulmonary baroreceptors has been thought to increase carotid baroreflex responsiveness in the quiescent state in man.

This microcirculatory study compared the effects on oxygen transport of two hemodilution fluids: HBOC-201 (Biopure Corp., Cambridge, MA) a Hemoglobin-Based Oxygen Carrier (HBOC), and 5.9% Human Serum Albumin (HSA) an iso-oncotic non-oxygen carrying colloid solution. Measurements using intravital microscopy were made on the spinotrapezius muscle of male, Sprague-Dawley rats. Interstitial PO2 was measured using phosphorescence quenching microscopy, and recorded before and after isovolemic hemodilutions (HD) at hematocrits of 40% (baseline), 30% (moderate HD) and 15% (severe HD). Oxygen consumption (VO2) of the spinotrapezius muscle was derived from PO2 recordings following the rapid inflation of a plastic bag placed around the objective. When the bag was inflated, blood flow in the muscle was arrested and PO2 rapidly fell over several seconds; the rate of decline of PO2 was proportional to VO2. For moderate HD (Hct ~ 30%) with HBOC-201, interstitial PO2 did not change from baseline conditions (Hct ~ 40%), while HD with HSA showed a decrease. For severe HD (Hct ~ 15%) both PO2 and VO2 were significantly lower for the HSA group than for the HBOC-201 group. These findings indicate that HBOC-201 maintains both a higher PO2 and VO2 during hemodiluted states compared with a non-oxygen carrying colloid solution (HSA). Furthermore, 5.9% HSA does not affect the mean arterial pressure (MAP) and vessel diameters, whereas HBOC-201 causes vasoconstriction, and consequently an increase in MAP. However, the vasoconstriction is not uniform among different branches of the arteriolar network, and most of the changes occur in the larger vessels, i.e., feed and arcade arterioles, while minimal in smaller vessels, i.e., transverse arterioles. In addition, findings show that MAP and vessel diameters return to baseline within 1-3 hours, implying that vasoconstriction and hypertension caused by HBOC-201 are acute responses.

Discoveries made with KCl-induced contractions have elucidated the more complex reactions involved in GPCRs signaling; once the mechanisms of smooth muscle Ca2+ sensitization and desensitization are fully understood, then the development of advanced treatments for vascular disorders such as hypertension, cerebral and coronary vasospasm, and vascular hyporeactivity following hemorrhagic shock may be possible. Studies have shown that KCl-induced contractions induce Ca2+-sensitization. Therefore, we tested the hypothesis that KCl induced Ca2+-sensitization is due to ROK activation by the increase in [Ca2+]i. To test this hypothesis, rabbit femoral arteries were permeabilized with 20µg/ml α-toxin and 1% Triton X-100 and subjected to different calcium concentrations in the presence or absence of various ROK inhibitors. For a comparison we also used various PKC and MLCK inhibitors and repeated these experiments in intact tissues. We found that either [Ca2+]i alone does not directly activate ROK or the permeabilization technique itself disrupts the normal ROK signaling system. Secondary findings revealed that α-toxin activates PKC pathways; in both chemically permeabilized preparations proteases also appear to be activated and MLCK is the primary kinase responsible for contraction.

Tonic arterial smooth muscle can produce strong contractions indefinitely by formation of slowly cycling crossbridges (latchbridges) that maintain force at a high energy economy. To fully understand the uniqueness of mechanisms regulating tonic arterial contraction, comparisons have been made to phasic visceral smooth muscles that do not sustain high forces. This study explored mechanisms of force maintenance in a phasic artery by comparing KCl-induced contractions in the tonic, femoral artery (FA) and its primary branch, the phasic saphenous artery (SA). KCl rapidly (5 N/m2) and [ca2+]i (250 nM) in FA and SA. By 10 min, [ca2+]i declined to 175 nM in both tissues but stress was sustained in FA (1.3 x 105N/m2) and reduced by 40% in SA (0.8 x l05 N/m2). Reduced tonic stress correlated with reduced myosin light chain (MLC) phosphorylation in SA (28% vs. 42% in FA). SA expressed more MLC phosphatase than FA, and permeabilized (β-escin) SA relaxed more rapidly than FA in the presence of MLC kinase blockade, suggesting that MLC phosphatase activity in SA was greater than that in FA. The reduction in MLC phosphorylation in SA was insufficient to account for reduced tonic force (latchbridge model), and SA expressed more "fast" myosin isoforms than did FA. Cytochalasin-D reduced force-maintenance more in FA than SA. These data support the hypothesis that strong force-maintenance is absent in SA because expressed motor proteins do not support latchbridge formation, and because actin polymerization is not stimulated.

It is well known that the overlap between the thick and thin filaments in striated muscle is responsible for the single active length-tension (L-T) curve. With the lack of visible striations, a sarcomeric unit has not been identified in smooth muscle. Though once thought to function like striated muscle via a sliding filament mechanism of contraction, recent studies on length-adaptation (L-adaptation) in airway smooth muscle (ASM), in which increased tension is generated with repeated contraction, have led to the hypothesis of a dynamic L-T curve in smooth muscle. Although more established in ASM, two studies have shown L-adaptation in vascular smooth muscle (VSM). In this project, the L-T curve over a 3-fold length range in rabbit femoral artery was investigated and the presence of more than one active and passive L-T curve was identified. The third of three repeated KCL-induced contractions at a single, shortened length resulted in L-adaptation in which the phasic and tonic phases of contraction demonstrated a 10-15% increase in active tension (Ta) relative to the first contraction. Experiments investigating possible mechanism(s) responsible for this phenomenon demonstrated that neither an increase in [Ca2+]i nor an increase in MLC20 phosphorylation was responsible for the increased tension. However, actin polymerization did appear to play a role in the L-adaptation of both phases of contraction. Thus directions for future research could include further study of actin polymerization in VSM that contributes to L-adaptation and may ultimately result in artery remodeling.