Philip J. Medeiros

Hypercapnic vs. hypoxic control of cardiovascular, cardiovagal, and sympathetic function

We compared the integrated cardiovascular and autonomic responses to hypercapnia and hypoxia to test the hypothesis that these stimuli differentially affect muscle sympathetic nerve activity (MSNA) discharge patterns and cardiovagal and sympathetic baroreflex function in a manner related to ventilatory chemoreflex sensitivity. Six males and six females underwent 5 min of hypoxia (end-tidal Po2 = 45 Torr) and 5 min of hypercapnia (end-tidal Pco2 = +8 Torr from baseline), causing similar ventilatory responses. A downward right shift in cardiovagal set point was observed during both conditions, which was strongly related to the change in inspiratory time (Ti) from baseline to hypercapnia (r2 = 0.67, P = 0.007) and hypoxia (r2 = 0.79, P < 0.001). Cardiovagal baroreflex gain was decreased during hypoxia (20.1 +/- 6.9 vs. 8.9 +/- 5.1 ms/mmHg, P < 0.001) but not hypercapnia (26.7 +/- 12.7 vs. 23.0 +/- 9.1 ms/mmHg). Both hypoxia and hypercapnia increased MSNA burst amplitude, whereas hypoxia, but not hypercapnia, also increased in MSNA burst frequency (21 +/- 9 vs. 28 +/- 7 bursts/min, P = 0.03) and total MSNA (4.56 +/- 3.07 vs. 7.37 +/- 3.26 mV/min, P = 0.002). However, neither hypercapnia nor hypoxia affected sympathetic burst probability or baroreflex gain. Hypoxia also caused a greater reduction in total peripheral resistance (P = 0.04), a greater increase in heart rate (P = 0.002), and a trend for a greater cardiac output response (P = 0.06) compared with hypercapnia. Nonetheless, central venous pressure remained unchanged during either condition. These results suggest that hypercapnia and hypoxia exert differential effects on cardiovagal, but not sympathetic, baroreflex gain and set point in a manner not related to ventilatory chemoreflex sensitivity. Furthermore, the data suggest that the individuals respiratory pattern to hypoxia or hypercapnia, as reflected in the inspiratory time, was a strong determinant of cardiovagal baroreflex set- point rather than the total ventilatory chemoreflex gain per se.

Neuropeptide Y stimulates proliferation and migration in the 4T1 breast cancer cell line

Stress has long been thought of to be associated with increased risk of cancer. Chronic stress is associated with elevated levels of sympathetic neurotransmitter (norepinephrine and neuropeptide Y: NPY) release and immunosuppression. The expression of NPY receptors has been reported in human breast carcinomas. Recently, activation of the NPY Y5 receptor was shown to stimulate cell growth and increase migration in human breast cancer cells; however the effects of NPY have yet to be investigated in a murine model of breast cancer. Thus, the specific aims of the current study were to: (i) characterize NPY receptor expression in 4T1 breast cancer cells and orthotopic tumors grown in BALB/c mice and (ii) investigate the impact of NPY receptor activation on 4T1 cell proliferation and migration in vitro. Positive expression of NPY receptors (Y1R, Y2R and Y5R) was observed in cells and tumor tissue. As well, NPY treatment of 4T1 cells promoted a concentration‐dependent increase in proliferation, through increased phosphorylation of ERK 1/2. Using NPY receptor antagonists (Y1R:BIBP3226, Y2R:BIIE0246 and Y5R:L‐152,804), we found the proliferative response to be Y5R mediated. Additionally, NPY increased chemotaxis through Y2R and Y5R activation. These data are in congruence with those from human cell lines and highlight the 4T1 cell line as a translatable model of breast cancer in which the effects of NPY can be studied in an immunocompetent system.

A Simple ''Streak Length Method'' for Quantifying and Characterizing Red Blood Cell Velocity Profiles and Blood Flow in Rat Skeletal Muscle Arterioles

Please cite this paper as: Al‐Khazraji BK, Novielli NM, Goldman D, Medeiros PJ, Jackson DN. A simple “Streak Length Method” for quantifying and characterizing red blood cell velocity profiles and blood flow in rat skeletal muscle arterioles. Microcirculation 19: 327–335, 2012.

Exercise training enhances insulin-stimulated nerve arterial vasodilation in rats with insulin-treated experimental diabetes

Insulin stimulates nerve arterial vasodilation through a nitric oxide (NO) synthase (NOS) mechanism. Experimental diabetes reduces vasa nervorum NO reactivity. Studies investigating hyperglycemia and nerve arterial vasodilation typically omit insulin treatment and use sedentary rats resulting in severe hyperglycemia. We tested the hypotheses that 1) insulin-treated experimental diabetes and inactivity (DS rats) will attenuate insulin-mediated nerve arterial vasodilation, and 2) deficits in vasodilation in DS rats will be overcome by concurrent exercise training (DX rats; 75-85% VO2 max, 1 h/day, 5 days/wk, for 10 wk). The baseline index of vascular conductance values (VCi = nerve blood flow velocity/mean arterial blood pressure) were similar (P ≥ 0.68), but peak VCi and the area under the curve (AUCi) for the VCi during a euglycemic hyperinsulinemic clamp (EHC; 10 mU·kg(-1)·min(-1)) were lower in DS rats versus control sedentary (CS) rats and DX rats (P ≤ 0.01). Motor nerve conduction velocity (MNCV) was lower in DS rats versus CS rats and DX rats (P ≤ 0.01). When compared with DS rats, DX rats expressed greater nerve endothelial NOS (eNOS) protein content (P = 0.04). In a separate analysis, we examined the impact of diabetes in exercise-trained rats alone. When compared with exercise-trained control rats (CX), DX rats had a lower AUCi during the EHC, lower MNCV values, and lower sciatic nerve eNOS protein content (P ≤ 0.03). Therefore, vasa nervorum and motor nerve function are impaired in DS rats. Such deficits in rats with diabetes can be overcome by concurrent exercise training. However, in exercise-trained rats (CX and DX groups), moderate hyperglycemia lowers vasa nervorum and nerve function.

Neuropeptide Y Y5-receptor activation on breast cancer cells acts as a paracrine system that stimulates VEGF expression and secretion to promote angiogenesis

Accumulating data implicate a pathological role for sympathetic neurotransmitters like neuropeptide Y (NPY) in breast cancer progression. Our group and others reported that NPY promotes proliferation and migration in breast cancer cells, however the angiogenic potential of NPY in breast cancer is unknown. Herein we sought to determine if NPY promotes angiogenesis in vitro by increasing vascular endothelial growth factor (VEGF) expression and release from 4T1 breast cancer cells. Western blot analysis revealed that NPY treatment caused a 52 ± 14% increase in VEGF expression in the 4T1 cells compared to non-treated controls. Using selective NPY Y-receptor agonists (Y1R, Y2R and Y5R) we observed an increase in VEGF expression only when cells were treated with Y5R agonist. Congruently, using selective Y1R, Y2R, or Y5R antagonists, NPY-induced increases in VEGF expression in 4T1 cells were attenuated only under Y5R antagonism. Endothelial tube formation assays were conducted using conditioned media (CM) from NPY treated 4T1 cells. Concentration-dependent increases in number of branch points and complete endothelial networks were observed in HUVEC exposed to NPY CM. CM from Y5R agonist treated 4T1 cells caused similar increases in number of branch points and complete endothelial networks. VEGF concentration was quantified in CM (ELISA) from agonist experiments; we observed a 2-fold and 2.5-fold increase in VEGF release from NPY and Y5R agonist treated 4T1 cells respectively. Overall these data highlight a novel mechanism by which NPY may promote breast cancer progression, and further implicate a pathological role of the NPY Y5R.

An automated cell-counting algorithm for fluorescently-stained cells in migration assays

A cell-counting algorithm, developed in Matlab®, was created to efficiently count migrated fluorescently-stained cells on membranes from migration assays. At each concentration of cells used (10,000, and 100,000 cells), images were acquired at 2.5 ×, 5 ×, and 10 × objective magnifications. Automated cell counts strongly correlated to manual counts (r2 = 0.99, P < 0.0001 for a total of 47 images), with no difference in the measurements between methods under all conditions. We conclude that our automated method is accurate, more efficient, and void of variability and potential observer bias normally associated with manual counting.

Neurogenic-nitric oxide interactions affecting brachial artery mechanics in humans: roles of vessel distensibility vs. diameter.

The purpose of this investigation was to assess the interactive influence of sympathetic activation and supplemental nitric oxide (NO) on brachial artery distensibility vs. its diameter. It was hypothesized that 1) sympathetic activation and NO competitively impact muscular conduit artery (brachial artery) mechanics, and 2) neurogenic constrictor input affects conduit vessel stiffness independently of outright changes in conduit vessel diastolic diameter. Lower body negative pressure (LBNP) and a cold pressor stress (CPT) were used to study the changes in conduit vessel mechanics when the increased sympathetic outflow occurred with and without changes in heart rate (LBNP -40 vs. -15 mmHg) and blood pressure (CPT vs. LBNP). These maneuvers were performed in the absence and presence of nitroglycerin. Neither LBNP nor CPT altered brachial artery diastolic diameter; however, distensibility was reduced by 25 to 54% in each reflex (all P < 0.05). This impact of sympathetic activation on brachial artery distensibility was not altered by nitroglycerin supplementation (21-54%; P < 0.05), although baseline diameter was increased by the exogenous NO (P < 0.05). The results indicate that sympathetic excitation can reduce the distensibility of the brachial artery independently of concurrent changes in diastolic diameter, heart rate, and blood pressure. However, exogenous NO did not minimize or reverse brachial stiffening during sympathetic activation. Therefore, sympathetic outflow appears to impact the stiffness of this conduit vessel rather than its diastolic diameter or, by inference, its local resistance to flow.

Pre-Diabetes Augments Neuropeptide Y1- and α1-Receptor Control of Basal Hindlimb Vascular Tone in Young ZDF Rats

Background Peripheral vascular disease in pre-diabetes may involve altered sympathetically-mediated vascular control. Thus, we investigated if pre-diabetes modifies baseline sympathetic Y1-receptor (Y1R) and α1-receptor (α1R) control of hindlimb blood flow (Qfem) and vascular conductance (VC). Methods Qfem and VC were measured in pre-diabetic ZDF rats (PD) and lean controls (CTRL) under infusion of BIBP3226 (Y1R antagonist), prazosin (α1R antagonist) and BIBP3226+prazosin. Neuropeptide Y (NPY) concentration and Y1R and α1R expression were determined from hindlimb skeletal muscle samples. Results Baseline Qfem and VC were similar between groups. Independent infusions of BIBP3226 and prazosin led to increases in Qfem and VC in CTRL and PD, where responses were greater in PD (p<0.05). The percent change in VC following both drugs was also greater in PD compared to CTRL (p<0.05). As well, Qfem and VC responses to combined blockade (BIBP3226+prazosin) were greater in PD compared to CTRL (p<0.05). Interestingly, an absence of synergistic effects was observed within groups, as the sum of the VC responses to independent drug infusions was similar to responses following combined blockade. Finally, white and red vastus skeletal muscle NPY concentration, Y1R expression and α1R expression were greater in PD compared to CTRL. Conclusions For the first time, we report heightened baseline Y1R and α1R sympathetic control of Qfem and VC in pre-diabetic ZDF rats. In support, our data suggest that augmented sympathetic ligand and receptor expression in pre-diabetes may contribute to vascular dysregulation.

Y1R control of sciatic nerve blood flow in the Wistar Kyoto rat

We hypothesized that neuropeptide Y (NPY) exerts vasoconstrictor properties in sciatic nerve blood supply by a Y1 receptor (Y1R) mechanism. Using Doppler ultrasound (40MHz), we measured blood flow velocity through a sciatic nerve supply artery during infusions of NPY and/or Y1R blockade with BIBP3226 in Wistar Kyoto rats before, and following, ganglionic blockade with Hexamethonium (Hex). Following Hex infusion, mean arterial pressure (baseline: 83±18, Hex: 57±3 mm Hg) was reduced. After 30 min, the index of conductance at the sciatic nerve (velocity/MAP expressed as % baseline) started to increase from 103±35 to 127±39% baseline in the following 30 min (p<0.05). Infusion of NPY (Y1 agonist) minimized this dilatory response (Hex baseline: 99±15, NPY: 104±11% baseline; NS). This NPY-induced attenuation was, in turn, minimized by BIBP3226 (Hex baseline: 73±12, NPY+BIBP3226: 89±14% baseline). Neither NPY nor BIBP3226 infusions without Hex affected the sciatic nerve arterial conductance. We conclude that the late dilation following Hex which is reversed by Y1R activation suggests some level of sympathetic control over sciatic nerve blood flow.