The Hemoglobin Homolog Cytoglobin in Smooth Muscle Inhibits Apoptosis and Regulates Vascular Remodeling

Abstract

Objective: The role of hemoglobin and myoglobin in the cardiovascular system is well established, yet other globins in this context are poorly characterized. Here, we examined the expression and function of cytoglobin (CYGB) during vascular injury.

Approach and results: We characterized CYGB content in intact vessels and primary vascular smooth muscle (VSM) cells and used 2 different vascular injury models to examine the functional significance of CYGB in vivo. We found that CYGB was strongly expressed in medial arterial VSM and human veins. In vitro and in vivo studies indicated that CYGB was lost after VSM cell dedifferentiation. In the rat balloon angioplasty model, site-targeted delivery of adenovirus encoding shRNA specific for CYGB prevented its reexpression and decreased neointima formation. Similarly, 4 weeks after complete ligation of the left common carotid, Cygb knockout mice displayed little to no evidence of neointimal hyperplasia in contrast to their wild-type littermates. Mechanistic studies in the rat indicated that this was primarily associated with increased medial cell loss, terminal uridine nick-end labeling staining, and caspase-3 activation, all indicative of prolonged apoptosis. In vitro, CYGB could be reexpressed after VSM stimulation with cytokines and hypoxia and loss of CYGB sensitized human and rat aortic VSM cells to apoptosis. This was reversed after antioxidant treatment or NOS2 (nitric oxide synthase 2) inhibition.

Conclusions: These results indicate that CYGB is expressed in vessels primarily in differentiated medial VSM cells where it regulates neointima formation and inhibits apoptosis after injury.

Keywords: angioplasty; apoptosis; arteriovenous fistula; myoglobin; nitric oxide.

Figure 1. Cytoglobin is expressed in human and rodent vessels

Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) results showing relative mRNA levels for cytoglobin (CYGB) and myoglobin (MB) in human aortas (n = 9, A), and human veins derived from vessels trimmings obtained from patients undergoing arteriovenous fistula (AVF) placement (n=26) or at revision (n=20) of failed AVFs (B). Levels of Cygb and Mb mRNA transcripts in mouse aorta (n=11), carotid (n = 6), and heart (n= 4, C); and rat aorta (n=5), carotid (n=3), and heart (n=6; D). Values represent the mean±SEM; *p≤0.05, compared to CYGB; **p≤0.01, compared to Cygb; ***p≤0.001, compared to Cygb. ****p≤0.0001, compared to Cygb.

Figure 2. Cytoglobin is expressed in medial SMCs and downregulated during de-differentiation in vitro and in vivo in the rat

A) qRT-PCR showing relative mRNA levels for Cygb, Myh11, and Lmod1 in the media of rat aorta (n=5) and subcultured medial SMCs (average from 4 different dispersions). B) Double immunofluorescence studies of rat carotids. We used antibodies against CYGB (green) and CNN1 (red). Blue staining is DAPI., yellow inset is shown at higher magnification on the lower right panel and shows colocalization (arrows) of CNN1 and CYGB in the media. A = adventitia, M = media, L = lumen. Representative of 3 independent experiments. C) WB analysis from medial rat aortic VSM cell at 0 or 120 hours after initial dispersion, and from subcultured RAVSMCs. Primary antibodies against CYGB, CNN1, and ACTA2 were used. Right panels, Densitometric analysis of results shown in left panel. Mean±SEM (n=3), *p≤0.05 and **p≤0.01 compared to 0 h. D) WB analysis showing the expression of CYGB in rat carotids following balloon injury in right uninjured (R) and left injured (L, INJ) carotids. Right panel, densitometric analysis. Values are mean ± SEM (n = 5-7). *P<0.05 compared with Sham, ***P<0.001 as compared to Sham, #P<0.05 as compared to 3 days. E) Left panels represent H&E staining of right uninjured (R) and left injured (L, INJ) carotids. Panel a, b, c, d, e, and f, double immunofluorescence staining of rat carotids, 14 days following balloon angioplasty injury. We used antibodies against CYGB (green) and CNN1. Blue staining is DAPI. A = adventitia, M = media, L = lumen. White dashed lines indicate the internal elastic lamina. Representative of 3 experiments.

Figure 3. Loss of Cytoglobin inhibits neointima formation in rodents

A) Western blot analysis showing the expression of CYGB in rat carotids from balloon-injured rat carotids treated with ShCon or ShCygb adenoviruses, 7 days post-injury and targeted adenoviral treatment. R indicates right uninjured carotid and L, Inj indicates left injured carotid. Values represent the mean ± SEM of the percentage change in CYGB immunoreactivity in left injured carotids relative to right non-injured carotids using ACTB as an internal reference; *P<0.05, n=5. B) representative H&E staining of left injured (L, Inj) carotids, 7 days after injury and targeted adenoviral delivery. Data shown is representative of 6 animals for each group. C) cross-sectional areas of the neointima, media, and lumen in rat carotids, 7 days after injury and targeted adenoviral delivery. Values shown represent the mean ± SEM (n=6). D) qRT-PCR showing relative mRNA levels for Cygb in mouse carotids of Cygb^+/+ and Cygb^-/- mice. Bars represent the Mean ± SEM (**P<0.01; n=5 and 6 for Cygb^+/+ and Cygb^{-/- ,} respectively). E) Representative H&E staining of cross-sections from Cygb^+/+ and Cygb^-/- mouse carotids. Rigt panel, morphological characterization of results shown in left panel. Bars represent the Mean ± SEM (n = 5). There was no statistically significant differences between Cygb^+/+ and Cygb^-/- mice. F) Neointima formation was induced by carotid artery ligation in Cygb^+/+ and Cygb^-/- mouse littermates and analyzed 4 weeks after ligation; Left panels are representative images of H&E staining of cross sections obtained from Cygb^+/+ and Cygb^-/- mice and morphological characterization are shown on the right panels, including medial, neointimal, and luminal areas, as well as external elastic lamina (EEL) circumference. There was no difference between Cygb^+/+ and Cygb^-/- mice except for neointimal areas. Points represent Mean ± SEM (n = 7, **P<0.01; ***P<0.001 vs. Cygb^+/+).

Figure 4. Loss of Cytoglobin exacerbates apoptosis in the injured vessel

A, WB showing the expression of CYGB in rat carotids from balloon-injured rat carotids treated with ShCon or ShCygb adenoviruses 4 days post-injury and targeted adenoviral treatment; n=4, *P<0.05. B, WB analysis showing the expression of PCNA in rat carotids from balloon-injured rat carotids, 4 days post-injury and targeted adenoviral treatment. Right panel, n.s. = not statistically significant. C, Representative results from immunohistochemistry analysis of carotids harvested from conditions as described in (A). Sections were stained for TUNEL. Arrows indicate occurrence of purple staining indicative of TUNEL; Analysis is shown on the right panel; n=4, *P<0.05 compared to shCon . D, and E, WB analysis showing the expression of pro- and cleaved caspase-3 (Panel D, n=7), and PARP (Panel E, arrows indicate occurrence of cleaved PARP, n = 4) in rat carotids harvested from conditions as described in (A). Graphs shown below each WB represent the mean ± SEM values derived from the densitometric analysis of WB. *P<0.01, **P<0.01, as determined by single or paired sample t-test with Bonferroni correction.

Figure 5. Increase in cell death upon loss of CYGB is O₂ and NO-dependent in rat aortic vascular smooth muscle

A) qRT-PCR results showing mRNA levels of CYGB in sub-cultured rat aortic vascular smooth muscle (RAVSM) cells. The cells were stimulated for 48 hours with interleukin-1β (IL-1β; 10 ng/ml) and interferon-γ (IFN-γ; 200 U/ml) at 21 or 1% O₂. Mean ± SEM, n = 8. B) WB results showing protein levels of CYGB in sub-cultured RAVSM using the same conditions described in (A). Densitometric analysis is shown on the right panel. Mean ± SEM, n ≥ 6; for (A) and (B) *P <0.05, **P<0.01, and ***P<0.001, as determined by single or paired sample t-test with Bonferroni correction. C) Western blot results showing protein levels of CYGB and NOS2 in rat aortic VSM cells in the presence of a cytokine mix (CM, IL-1β + IFN-γ) with or without adenoviral silencing of CYGB.. Densitometric analysis is shown on the right panels. Mean ± SEM, n = 4;*P <0.05, **P<0.01, as determined by paired sample t-test with Bonferroni correction. D) Cells were stimulated with IL-1β and IFN-γ for 48 hours at 21 or 1% O₂ with or without silencing of CYGB and cytotoxicity was determined by measuring LDH release. Mean ± SEM, n =3 E) cells were stimulated with IL-1β and IFN-γ for 48 hours at 21% O₂ with or without silencing of CYGB and incubated with staurosporine for 24 hours before cytotoxicity was determined by measuring LDH release. Mean ± SEM, n =5. for (D) and (E) **P<0.01, and ***P<0.001, as determined by 2-way ANOVA.

Figure 6. CYGB inhibits staurosporine induced apoptosis in human aortic SMCs independent of cell proliferation

A) qRT-PCR results showing relative mRNA levels for CYGB, MYH11, and LMOD1 in six human aortas and sub-cultured human aortic smooth muscle cells (VSMCs) derived from 3 different dispersions. B) Sub-cultured human aortic SMCs were electroporated with siRNA mix targeting CYGB (SiCYGB) or scrambled siRNA (Scr). Western blots show protein levels of CYGB, NOS2, and NOS1. Values represent the mean ± SEM of the percentage change in CYGB immunoreactivity compared to non-transfected conditions (Control) using ACTB as an internal reference; n = 4, *P<0.05 C) Human Aortic VSMCs were incubated with serum or serum + PDGF for 3 days and cell count was determined. There was no difference between SiCYGB and Scr treated cells. Mean ± SEM with n = 4. D) Sub-cultured human aortic SMCs were treated with staurosporine (STS, 1 μM) for 24 hours in the presence or absence of the pan-caspase inhibitor (z-VAD-Fmk) or N-acetyl cysteine (NAC). Cytotoxicity was determined by measuring LDH release. Mean ± SEM, with n = 4-6. *P<0.05. E) Western blot analysis for Caspase-3 (Pro-CASP3) and cleaved CASP3 from human aortic SMCs incubated with staurosporine (1 μM for 6 hours) showing increased in cleaved caspase-3 upon silencing of CYGB. Values represent the mean ± SEM of the percentage change in cleaved CASP3 immunoreactivity in SiCYGB and Scr treated cells compared to non-transfected conditions(Control) using ACTB as an internal reference; n = 3, *P<0.05.