Modulating Vascular Hemodynamics With an Alpha Globin Mimetic Peptide (HbαX)

Abstract

The ability of hemoglobin to scavenge the potent vasodilator nitric oxide (NO) in the blood has been well established as a mechanism of vascular tone homeostasis. In endothelial cells, the alpha chain of hemoglobin (hereafter, alpha globin) and endothelial NO synthase form a macromolecular complex, providing a sink for NO directly adjacent to the production source. We have developed an alpha globin mimetic peptide (named HbαX) that displaces endogenous alpha globin and increases bioavailable NO for vasodilation. Here we show that, in vivo, HbαX administration increases capillary oxygenation and blood flow in arterioles acutely and produces a sustained decrease in systolic blood pressure in normal and angiotensin II-induced hypertensive states. HbαX acts with high specificity and affinity to endothelial NO synthase, without toxicity to liver and kidney and no effect on p50 of O₂ binding in red blood cells. In human vasculature, HbαX blunts vasoconstrictive response to cumulative doses of phenylephrine, a potent constricting agent. By binding to endothelial NO synthase and displacing endogenous alpha globin, HbαX modulates important metrics of vascular function, increasing vasodilation and flow in the resistance vasculature.

Keywords: alpha globin; blood pressure; endothelial nitric oxide synthase; endothelium; mimetic peptide.

Figure 1. Acute increased O₂ saturation and blood flow in microvasculature with single injection of HbαX in vivo

(A–D) Photoacoustic microscopy images of O₂ saturation (sO₂) and blood flow from microvasculature of murine ear at baseline (A–B) and 120 minutes post HbαX injection (C–D). All images are from the same portion of the ear. The ^ symbol denotes an arteriole and # denotes a venule. Qualitatively, sO₂ increases after HbαX injection shown by warmer colors in the regions of capillary beds. Blood flow is shown in both arterioles and venules with red coloring denoting regions of highest flow. Quantification of sO₂ (E) and average flow velocity (F) from mice receiving HbαX injection (solid lines, n=3) and saline injection (dotted lines, n=3); red denotes arteriolar value, blue denotes venular. Scale bar in A is 100 μm.

Figure 2. Sustained decrease in systolic blood pressure with HbαX

Either normotensive mice (A: N=4) or mice with osmotic pumps loaded with AngII (B: N=4) were injected every other day starting at day 0 with saline (black), a scrambled control peptide (green; 5 mg/kg) or HbαX (blue; 5 mg/kg) and systolic blood pressure via radiotelemetery was obtained daily. A baseline was measured for 10 days prior to the start of injections. Red line in B denotes approximate end of AngII infusion due osmotic pump lifetime. Superoxide production from kidney (C) and mesenteric (D) tissues was assayed from mice with AngII pumps and HbαX injections (n=3) or saline injections (n=3), or receiving sham surgery (n=4).

Figure 3. Prolonged injection of the alpha globin mimetic peptide does not lead to murine toxicity

Approximately 200 μL of blood was drawn via submandibular puncture every 15 days, starting prior to the first injections on day 0 of saline (black), control peptide (green) or HbαX (blue). The blood was subject to blood pathology laboratories at the University of Virginia to test for basic parameters that could indicate toxicity. These include a change in levels of hemoglobin (A), white blood cells in aggregate (B), urea (C), ALT (D), AST (E), and creatinine (F). Although some variability was observed, there was little significant difference using two-way ANOVA and an alpha level of either p<0.05 or p<0.10. N=3 mice per condition.

Figure 4. An alpha globin mimetic peptide and eNOS dimers bind with high affinity

(A) Co-immunoprecipitation of eNOS and biotin-labeled HbαX from hAoECs shows that HbαX binds to the dimeric form of eNOS. Non-denaturing IP and electrophoresis conditions were used to preserve the conformational state of eNOS. Lane 1 shows EC lysate as a control for eNOS species. Lane 2 is from beads incubated with EC Lysate and biotin-conjugated HbαΧ peptide. Lane 3 is sample eluted from dynabeads with low pH. (B) Fluorescence polarization assays show nanomolar binding affinity between fluorescein isothiocyanate (FITC) – labeled HbαX and the oxygenase domain of eNOS. The concentration of FITC-HbαX is 5 nmol/L in (B). The total concentration of peptide in (C) was held constant, but only half (2.5 nmol/L) was fluorescently tagged. This effectively will double the observed binding affinity because of the 1:1 competition for binding site. (D) shows no curve because no binding was observed between HbαX peptides. The concentration of labeled peptide was held constant (5 nmol/L) and was used to serially dilute unlabeled HbαX. In B–D, the points represent mean and bars indicate standard deviation, samples were run in triplicate in one experimental preparation.

Figure 5. An alpha globin mimetic peptide does not alter the O₂ binding parameters of hemoglobin

(A) The oxygen p50 of whole human blood was measured and was found not to be altered by incubation for 5 minutes with HbαX (blue) or a scrambled control peptide (green) (n=3 experiments for each condition). (B) Fluorescence polarization of 5 nmol/L FITC-labeled HbαX incubated with bovine hemoglobin shows no concentration-dependent binding of the molecules due to the lack of a hyperbolic shape.

Figure 6. An alpha globin mimetic peptide significantly reduces hyperconstriction in arterioles from human patients with hypertension

(A) Far-Western blotting for recombinant human eNOS oxygenase domain on a non-denaturing gel using anti-eNOS and biotin-HbαX probes and corresponding secondary antibodies for fluorescent detection. Left lane serves as a molecular weight marker, but it is not used to estimate molecular weight due to the non-denaturing condition of the gel. The samples were run in duplicate. (B) Western blot of the expression of alpha globin without its beta globin partner is confirmed in human arterioles, and a 10-fold dilution of RBC samples is used as a positive control for alpha and beta globin chains. Note the lack of alpha globin in aorta. Arrowheads for CD31 indicate 100 kDa, and alpha and beta globin indicate 10 kDa. (C) Resistance arterioles (approximately 100 μm in diameter) isolated from human adipose tissue biopsies were cannulated and subject to phenylephrine cumulative dose-response curves in the absence (black) or presence (blue) of 5 μmol/L HbαX. (n=4). * indicates p<0.05, ** indicates p<0.01 using two-way ANOVA and a Bonferroni post-test.