A flow cytometric method for the analysis of macrophages in the vascular wall.

Macrophages accumulate in the vascular wall during conditions such as hypertension and hypercholesterolemia and contribute to vascular remodelling. Here we describe a method for the isolation and subsequent flow cytometric analysis of macrophages from aortas of mice. Cell suspensions were prepared from thoracic aortas of male C57BL/6J mice by a combination of manual disruption, incubation in enzymatic digestion medium, and passage through a 70 µm cell strainer. Flow cytometric analysis of these suspensions revealed a high content of cells with strong light scattering properties (i.e. SSC(hi)) compared with suspensions derived from mouse blood, spleen, thymus or kidney. Unstained aortic cell suspensions also displayed a preponderance of autofluorescence in the B670, V560, V460, B525 and V610 channels of the flow cytometer, suggesting that these channels should be avoided for subsequent flow cytometric analyses, at least for initial gating steps. Thus, aortic preparations were labelled with an APC-Cy7-conjugated antibody against the pan-leukocyte marker, CD45, as well as an APC-conjugated antibody against the macrophage-specific antigen, F4/80, as these fluorochromes emit in channels that displayed relatively low levels of auto-fluorescence in our initial studies (i.e. R780 and R660). Flow cytometric analysis of labelled aortic preparations revealed a distinct population of CD45(+)F4/80(+) cells. Importantly, back-gating on this CD45(+)F4/80(+) cell population showed it to be now virtually devoid of autofluorescence in all remaining open channels, and hence an appropriate foundation for further detailed analysis of macrophage polarization using multiple intra- and extra-cellular markers. Furthermore, we demonstrated that angiotensin II-induced hypertension in C57BL6/J mice, and hypercholesterolemia in apolipoprotein E-deficient mice, each resulted in an approximate doubling of CD45(+)F4/80(+) cells in the aortic wall, highlighting the utility of our new protocol for studying the impact of disease on macrophage accumulation in the vascular wall.

Nitroxyl (HNO) suppresses vascular Nox2 oxidase activity.

Nox2 oxidase activity underlies the oxidative stress and vascular dysfunction associated with several vascular-related diseases. We have reported that nitric oxide (NO) decreases reactive oxygen species production by endothelial Nox2. This study tested the hypothesis that nitroxyl (HNO), the redox sibling of NO, also suppresses vascular Nox2 oxidase activity. Specifically, we examined the influence of two well-characterized HNO donors, Angeli's salt and isopropylamine NONOate (IPA/NO), on Nox2-dependent responses to angiotensin II (reactive oxygen species production and vasoconstriction) in mouse cerebral arteries. Angiotensin II (0.1µmol/L)-stimulated superoxide (measured by lucigenin-enhanced chemiluminescence) and hydrogen peroxide (Amplex red fluorescence) levels in cerebral arteries (pooled basilar and middle cerebral (MCA)) from wild-type (WT) mice were ~60% lower (P<0.05) in the presence of either Angeli's salt (1µmol/L) or IPA/NO (1µmol/L). Similarly, phorbyl 12,13-dibutyrate (10µmol/L; Nox2 activator)-stimulated hydrogen peroxide levels were ~40% lower in the presence of IPA/NO (1µmol/L; P<0.05). The ability of IPA/NO to decrease superoxide levels was reversible and abolished by the HNO scavenger l-cysteine (3mmol/L; P<0.05), but was unaffected by hydroxocobalamin (100µmol/L; NO scavenger), ODQ (10µmol/L; soluble guanylyl cyclase (sGC) inhibitor), or Rp-8-pCPT-cGMPS (10µmol/L; cyclic guanosine monophosphate (cGMP)-dependent protein kinase inhibitor). Angiotensin II-stimulated superoxide was substantially less in arteries from Nox2-deficient (Nox2(-/y)) versus WT mice (P<0.05). In contrast to WT, IPA/NO (1µmol/L) had no effect on superoxide levels in arteries from Nox2(-/y) mice. Finally, angiotensin II (1-1000µmol/L)-induced constriction of WT MCA was virtually abolished by IPA/NO (1µmol/L), whereas constrictor responses to either the thromboxane A2 mimetic U46619 (1-100 nmol/L) or high potassium (122.7mmol/L) were unaffected. In conclusion, HNO suppresses vascular Nox2 oxidase activity via a sGC-cGMP-independent pathway. Thus, HNO donors might be useful therapeutic agents to limit and/or prevent Nox2-dependent vascular dysfunction.