Michael C. Madigan

Therapeutic Potential of the Nitrite-Generated NO Pathway in Vascular Dysfunction

Nitric oxide (NO) generated through L-arginine metabolism by endothelial nitric oxide synthase (eNOS) is an important regulator of the vessel wall. Dysregulation of this system has been implicated in various pathological vascular conditions, including atherosclerosis, angiogenesis, arteriogenesis, neointimal hyperplasia, and pulmonary hypertension. The pathophysiology involves a decreased bioavailability of NO within the vessel wall by competitive utilization of L-arginine by arginase and “eNOS uncoupling.” Generation of NO through reduction of nitrate and nitrite represents an alternative pathway that may be utilized to increase the bioavailability of NO within the vessel wall. We review the therapeutic potential of the nitrate/nitrite/NO pathway in vascular dysfunction.

Nuclear matrix localization of high mobility group protein I(Y) in a transgenic mouse model for prostate cancer

Nuclear shape and the underlying nuclear structure, the nuclear matrix in cancer cells. Since the NM composition is considered to maintain nuclear shape and architecture, nuclear matrix proteins (NMPs) may be involved in transformation. Our laboratory has recently characterized a subset of NMPs that are associated with prostate cancer development in the transgenic adenocarcinoma of mouse prostate (TRAMP) model. One of the identified NMPs, E3E, has a similar molecular weight (22 kDa) with a protein known as HMGI(Y). HMGI(Y) belongs to a group of non‐histone and chromatin‐associated proteins, high‐mobility‐group (HMG) proteins, and it has been shown to associate with the NM. HMGI(Y) has been reported to be elevated in different types of cancer including prostate cancer. In this study, we examined the expression of HMGI(Y) protein in the NMP composition of the TRAMP model during the progression from normal to neoplasia. The expression of HMGI(Y) in the NMP extracts of three prostatic epithelial cell lines derived from a 32‐week TRAMP mouse: TRAMP‐C1, TRAMP‐C2, and TRAMP‐C3 was also examined. Using both one‐dimensional and high‐resolution two‐dimensional immunoblot analyses, we found that: (i) HMGI(Y) is a nuclear matrix protein expressed as two protein bands with MW of 22–24 kDa and (ii) HMGI(Y) expression is correlated with neoplastic and malignant properties in late stage TRAMP prostate tumors. Overall, these findings support the evidence that HMGI(Y) can be utilized as a marker and prognostic tool for prostate cancer.

Xanthine Oxidoreductase Function Contributes to Normal Wound Healing.

Chronic, nonhealing wounds result in patient morbidity and disability. Reactive oxygen species (ROS) and nitric oxide (NO) are both required for normal wound repair, and derangements of these result in impaired healing. Xanthine oxidoreductase (XOR) has the unique capacity to produce both ROS and NO. We hypothesize that XOR contributes to normal wound healing. Cutaneous wounds were created in C57Bl6 mice. XOR was inhibited with dietary tungsten or allopurinol. Topical hydrogen peroxide (H2O2, 0.15%) or allopurinol (30 µg) was applied to wounds every other day. Wounds were monitored until closure or collected at d 5 to assess XOR expression and activity, cell proliferation and histology. The effects of XOR, nitrite, H2O2 and allopurinol on keratinocyte cell (KC) and endothelial cell (EC) behavior were assessed. We identified XOR expression and activity in the skin and wound edges as well as granulation tissue. Cultured human KCs also expressed XOR. Tungsten significantly inhibited XOR activity and impaired healing with reduced ROS production with reduced angiogenesis and KC proliferation. The expression and activity of other tungsten-sensitive enzymes were minimal in the wound tissues. Oral allopurinol did not reduce XOR activity or alter wound healing but topical allopurinol significantly reduced XOR activity and delayed healing. Topical H2O2 restored wound healing in tungsten-fed mice. In vitro, nitrite and H2O2 both stimulated KC and EC proliferation and EC migration. These studies demonstrate for the first time that XOR is abundant in wounds and participates in normal wound healing through effects on ROS production.

Delayed inhaled carbon monoxide mediates the regression of established neointimal lesions

OBJECTIVE Intimal hyperplasia (IH) contributes to the failure of vascular interventions. While many investigational therapies inhibit the development of IH in animal models, few of these potential therapies can reverse established lesions. Inhaled carbon monoxide (CO) dramatically inhibits IH in both rats and pigs when given perioperatively. It also prevented the development of pulmonary arterial hypertension in rodents. Interestingly, CO could reverse pulmonary artery structural changes and right heart hemodynamic changes when administered after the establishment of pulmonary hypertension. Thus, we hypothesize that inhaled CO may mediate the regression of established neointimal lesions. METHODS Rats underwent carotid artery balloon angioplasty injury. Carotid arteries were collected at 2 and 4 weeks after injury for morphometric analysis of the neointima. Another group was treated with inhaled CO (250 parts per million) for 1 hour daily from week 2 until week 4. Additional rats were sacrificed 3 days after initiating CO treatment, and the carotid arteries were examined for apoptosis by terminal deoxynucleotidyl transferase dUTP nick end-labeling, proliferation by Ki67 staining, and autophagy by microtubule-associated protein light chain 3 I/II staining. RESULTS At 2 weeks following injury, sizable neointimal lesions had developed (intimal/media = 0.92 ± 0.22). By 4 weeks, lesion size remained stable (0.80 ± 0.09). Delayed inhaled CO treatment greatly reduced neointimal lesion size vs the 2- and 4-week control mice (0.38 ± 0.05; P < .05). Arteries from the CO-treated rats exhibited significantly reduced apoptosis compared with control vessels (3.18% ± 1.94% vs 16.26% ± 5.91%; P = .036). Proliferation was also dramatically reduced in the CO-treated animals (2.98 ± 1.55 vs 10.37 ± 2.80; P = .036). No difference in autophagy between control and CO-treated rats was detected. CONCLUSIONS Delayed administration of inhaled CO reduced established neointimal lesion size. This effect was mediated by the antiproliferative effect of CO on medial and intimal smooth muscle cells without increases in arterial wall apoptosis or autophagy. Future studies will examine additional time points to determine if there is temporal variation in the rates of apoptosis and autophagy.

P2Y2 nucleotide receptor mediates arteriogenesis in a murine model of hind limb ischemia

OBJECTIVE Arteriogenesis represents the maturation of preformed vascular connections in response to flow changes and shear stress. These collateral vessels can restore up to 60% of the native blood flow. Shear stress and vascular injury can induce the release of nucleotides from vascular smooth muscle cells and platelets that can serve as signaling ligands, suggesting they may be involved in mediating arteriogenesis. The P2Y2 nucleotide receptor (P2Y2R) has also been shown to mediate smooth muscle migration and arterial remodeling. Thus, we hypothesize that P2Y2R mediates arteriogenesis in response to ischemia. METHODS Hind limb ischemia was induced by femoral artery ligation (FAL) in C57Bl/6NJ or P2Y2R negative mice (P2Y2(-/-)). Hind limb perfusion was measured with laser Doppler perfusion imaging and compared with the sham-operated contralateral limb immediately and at 3, 7, 14, 21, and 28 days after ligation. Collateral vessel size was measured by Microfil casting. Muscle specimens were harvested and analyzed with immunohistochemistry for Ki67, vascular cell adhesion molecule, macrophages, and muscle viability by hematoxylin and essoin stain. RESULTS Hind limb ischemia induced by FAL in C57Bl/6NJ mice resulted in significant ischemia as measured by laser Doppler perfusion imaging. There was rapid recovery to nearly normal levels of perfusion by 2 weeks. FAL in P2Y2(-/-) mice resulted in severe ischemia with greater tissue loss. Recovery of perfusion was impaired, achieving only 40% compared with wild-type mice by 28 days. Collateral vessels in the P2Y2(-/-) mice were underdeveloped, with reduced vascular cell proliferation and smaller vessel size. The collaterals were ∼65% the size of wild-type collateral vessels (P = .011). Angiogenesis at 28 days in the ischemic muscle, however, was greater in the P2Y2(-/-) mice (P < .001), possibly related to persistent ischemia leading and angiogenic drive. Early macrophage recruitment was reduced by nearly 70% in P2Y2(-/-) despite significantly more myocyte necrosis. However, inflammation was greater at 28 days in the P2Y2(-/-) mice. CONCLUSIONS P2Y2R deficiency does not alter baseline collateral vessel formation but does significantly impair collateral maturation, with resultant persistent limb ischemia despite enhanced angiogenesis. These findings reinforce the importance of arteriogenesis in the recovery of perfusion in ischemic tissues compared with angiogenesis. They also support the role of P2Y2R in mediating this process. The mechanism by which P2Y2R mediates arteriogenesis may involve the recruitment of inflammatory cells to the ischemic tissues, which is essential to arteriogenesis. Approaches to target P2Y2R may yield new therapeutic strategies for the treatment of arterial occlusive disease.

Occult type I or III endoleaks are a common cause of failure of type II endoleak treatment after endovascular aortic repair

Objective Most type II endoleaks have a benign natural history, but 6% to 8% are associated with sac enlargement and respond poorly to treatment. Our aim was to evaluate whether these enlargements are associated with delayed or occult type I and III endoleaks. Methods Patients with interventions for endoleak after endovascular aortic repair from 2000 to 2016 were reviewed retrospectively. Patient demographics, comorbidities, endoleak type, secondary procedures, aortic sac growth (≥5 mm), and mortality were collected. Successful treatment was defined as endoleak resolution with no further aortic sac growth. Secondary procedures, ruptures, endograft explant, and death were captured. Results There were 130 patients diagnosed with a primary type II endoleak after endovascular aortic repair at a median of 1.3 months (interquartile range, 1.0‐13.3 months). One hundred eighteen had their initial treatment for a primary type II. Twelve of the 130 were initially stable and observed, but were treated for a delayed type I or III endoleak. The 130 patients underwent 279 procedures for endoleaks (mean of 2.2 ± 1.3) over 6.9 ± 3.8 years of follow‐up. Of the 118 patients treated for primary type II endoleaks, 26 (22.0%) later required interventions for delayed type I and III endoleaks. The mean time to intervention for a delayed type I or III endoleak was 5.4 ± 2.8 years. Overall, there were 16 type IA, 11 type IB, 2 type III, 7 combined type IA/IB, and 2 type IA/III delayed endoleaks. The odds of harboring a delayed type I or III endoleak was 22.0% before the first attempt at type II endoleak treatment, 35.1% before the second, 44.8% before the third, and 66.6% before the fourth attempts. Rapid aortic sac growth of ≥5 mm/y before initial endoleak treatment was associated with increased risk for delayed type I or III endoleak (47.8 vs 14.1%; P = .003). Patients with delayed type I or III endoleaks had a lower successful treatment rate (8.3% vs 52.3%; P = .001) than those with only type II endoleaks. Late rupture was increased with delayed type I or III endoleak (P = .002), whereas mortality (P = .96) and aortic‐related mortality (P = .46) were similar. Graft explant (P = .06) trended toward an increase with a delayed type I or III endoleak, but was not statistically significant. Conclusions Failed attempts treating type II endoleaks and/or a rapid aortic sac growth of 5 mm/y or greater should raise the suspicion of a delayed or occult type I or III endoleak. Occult endoleaks are associated with decreased chance of endoleak resolution.