Katušić, Zvonimir S.

Evidence continues to accumulate on the importance of endothelial progenitor cells (EPCs) in angiogenesis and repair of injured vascular endothelium. Elevated expression of key antioxidant enzymes and their ability to inactivate reactive oxygen species appears to be responsible for resistance of EPCs to oxidative stress. “Late” (outgrowth) EPCs express high levels of manganese superoxide dismutase (MnSOD) enabling them to maintain morphological and functional integrity of mitochondria under conditions associated with increased local concentration of superoxide anions. In “early” EPCs expression of catalase, glutathione peroxidase (GPx), and MnSOD are significantly higher as compared to mature endothelium thereby increasing their resistance to cytotoxic effects not only of superoxide anions but to hydrogen peroxide as well. In addition, in vitro studies demonstrated that EPCs are capable of executing regenerative program even when exposed to pro‐oxidant environment. Consistent with these observations, in vivo genetic inactivation of GPx‐1 in mouse causes profound impairment of regenerative function of EPCs demonstrating that oxidative stress has detrimental effect on EPCs with disrupted antioxidant defense system. Further studies are required to precisely define the role of individual reactive oxygen species and antioxidant enzymes in control of regenerative function of EPCs.

The mechanisms underlying regenerative function of endothelial progenitor cells (EPCs) are not completely understood. Since previous studies from several groups including ours suggest that biosynthesis of NO in EPCs is low, the present study was designed to determine ability of EPCs to produce and release prostacyclin (PGI 2 ). Outgrowth (“late”) EPCs were obtained from peripheral blood of healthy subjects. Enzymatic activity of eNOS was significantly lower in EPCs as compared to eNOS activity in human coronary endothelial cells (CAECs) (n=4‐6 subjects, P<0.05). In contrast, production and release of PGI 2 was four fold higher in EPCs as compared to CAECs (n=3, P<0.05). This was associated with significantly higher expression of cyclooxygenase‐1 (COX‐1) protein in EPCs whereas levels of COX‐2 protein were not different between progenitor and mature endothelial cells. Production of thromboxane A 2 and prostaglandin E 2 were low and identical in EPCs and CAECs. Most importantly, inhibition of COX isoforms with indomethacin impaired pro‐angiogenic effect of EPCs as assessed by in vitro tube formation assay (n=4‐6, P<0.05). Cell proliferation was also significantly reduced after inhibition of COX isoforms with indomethacin or inactivation of PGI 2 ‐synthase by siRNA (n=4‐6, P<0.05). Our findings suggest that production and release PGI 2 is an important mechanism contributing to regenerative capability of EPCs.

Heme oxygenase (HO) activity is exhibited by inducible (HO-1) and constitutive (HO-2) proteins. HO-1 protects against ischemic and nephrotoxic acute kidney injury (AKI). We have previously demonstrated that HO-2 protects against heme protein-induced AKI. The present study examined whether HO-2 is protective in ischemic AKI. Renal ischemia was imposed on young and aged HO-2+/+ and HO-2−/− mice. On days 1 and 2 after renal ischemia, there were no significant differences in renal function between young male HO-2+/+ and HO-2−/− mice, between young female HO-2+/+ and HO-2−/− mice, or between aged female HO-2+/+ and HO-2−/− mice. However, in aged male mice, HO-2 deficiency worsened renal function on days 1 and 2 after ischemic AKI, and, on day 2 after ischemia, such deficiency augmented upregulation of injury-related genes and worsened histological injury. Renal HO activity was markedly decreased in unstressed aged male HO-2−/− mice and remained so after ischemia, despite exaggerated HO-1 induction in HO-2−/− mice after ischemia. Such exacerbation of deficiency of HO-2 protein and HO activity may reflect phosphorylated STAT3, as activation of this proinflammatory transcription factor was accentuated early after ischemia in aged male HO-2−/− mice. This exacerbation may not reflect impaired induction of nephroprotectant genes, since the induction of HO-1, sirtuin 1, and β-catenin was accentuated in aged male HO-2−/− mice after ischemia. We conclude that aged male mice are hypersensitive to ischemic AKI and that HO-2 mitigates such sensitivity. We speculate that this protective effect of HO-2 may be mediated, at least in part, by suppression of phosphorylated STAT3-dependent signaling.

The amyloid precursor protein (APP) is expressed in the blood vessel wall, but the physiological function of APP is not completely understood. Previous studies established that APP has amine oxidase activity responsible for degradation of catecholamines. In the present study, we characterized the vascular phenotype of APP-knockout (APP−/−) mice. We demonstrate that circulating levels of catecholamines are significantly increased in male as compared with female APP−/− mice. Studies of vasomotor function in isolated aortas revealed that contractions to the α1-receptor agonist phenylephrine were significantly reduced in male APP−/− mice but not in females. In addition, contractions to G protein activation with sodium fluoride were reduced exclusively in male APP−/− mice aortas. The endothelium-dependent relaxations to acetylcholine were not affected by the loss of APP in mice of both sexes. Further analysis of the mechanisms underlying endothelium-dependent relaxations revealed that inhibition of cyclooxygenase by indomethacin significantly impaired relaxations to acetylcholine exclusively in male APP−/− mice. Furthermore, acetylcholine-induced production of cyclic guanosine monophosphate (cGMP) was significantly reduced in male APP−/− mice aortas while acetylcholine-induced production of cyclic adenosine monophosphate (cAMP) was enhanced. We concluded that altered vascular reactivity to phenylephrine appears to be in part the result of chronic exposure of male APP−/− aorta to high circulating levels of catecholamines. The mechanisms responsible for the impairment of endothelium-dependent cGMP signaling and adaptive enhancement of endothelium-dependent production of cAMP remain to be defined. NEW & NOTEWORTHY Male amyloid precursor protein (APP)-deficient mice have higher circulating levels of catecholamines as compared with female APP-deficient mice. As a consequence, endothelium-dependent and endothelium-independent vasomotor functions of male APP-deficient mice are significantly altered. Under physiological conditions, expression of APP appears to play an important role in vascular function.

Abstract Increasing evidence recognizes Alzheimer's disease (AD) as a multifactorial and heterogeneous disease with multiple contributors to its pathophysiology, including vascular dysfunction. The recently updated AD Research Framework put forth by the National Institute on Aging–Alzheimer's Association describes a biomarker‐based pathologic definition of AD focused on amyloid, tau, and neuronal injury. In response to this article, here we first discussed evidence that vascular dysfunction is an important early event in AD pathophysiology. Next, we examined various imaging sequences that could be easily implemented to evaluate different types of vascular dysfunction associated with, and/or contributing to, AD pathophysiology, including changes in blood‐brain barrier integrity and cerebral blood flow. Vascular imaging biomarkers of small vessel disease of the brain, which is responsible for >50% of dementia worldwide, including AD, are already established, well characterized, and easy to recognize. We suggest that these vascular biomarkers should be incorporated into the AD Research Framework to gain a better understanding of AD pathophysiology and aid in treatment efforts.

Cerebral amyloid angiopathy (CAA) is present in over half of the elderly population and in 80–90% of Alzheimer’s disease (AD) patients. CAA is defined by the deposition of beta amyloid (Aβ) in small cerebral arteries and capillaries. Cardiovascular risk factors are associated with an increased incidence of CAA. We utilized 18-month-old endothelial nitric oxide synthase (eNOS) heterozygous knockout ( +/− ) mice, a clinically relevant model of endothelial dysfunction, to examine the role of endothelial nitric oxide (NO) in vascular Aβ accumulation. eNOS +/− mice had significantly higher vascular levels of Aβ40 ( P < 0.05). Aβ42 was not detected. There was no difference in Aβ in brain tissue. Amyloid precursor protein and β-site APP cleavage enzyme 1 protein levels were unaltered, while levels of the α-secretase enzyme, a disintegrin and metalloproteinase 10, were significantly lower in eNOS + /− microvascular tissue ( P < 0.05). Insulin degrading enzyme and low-density lipoprotein receptor-related protein 1 were significantly increased in eNOS +/− microvascular tissue, most likely an adaptive response to locally higher Aβ concentrations. Lastly, catalase and CuZn superoxide dismutase were significantly elevated in eNOS +/− microvascular tissue ( P < 0.05). These data demonstrate decreased availability of endothelial NO leads to increased cerebrovascular concentration of Aβ along with compensatory mechanisms to protect the vasculature.

Amyloid precursor protein (APP) is an integral membrane protein expressed in the peripheral arteries. However, the exact vascular physiological function of APP is unknown. Male APP-deficient (APP –/– ) and their wild-type littermates (WT) mice were used to characterize the phenotype of APP in the control of vascular function. Isometric force of isolated aortic rings was recorded in organ chambers. Circulating levels of norepinephrine and epinephrine were significantly enhanced in APP –/– mice (4723±566 pg/mL and 854±98 pg/mL, respectively P<0.05 vs. WT: 1999±319 pg/mL and 429±71 pg/mL, respectively; n=13). The efficacy of phenylephrine induced contractions were significantly reduced in the aorta of APP –/– mice (21±3%, P<0.05 vs. WT: 47±4%; n=10) while contractions to prostaglandin F 2α were unchanged (135±4%, P=n.s. vs. WT: 133±3%; n=9). Western blot analysis revealed that protein expression of alpha1D adrenergic receptors was significantly downregulated in APP –/– mice aortas (0.21±0.05 O.D.; P<0.05 vs. WT: 0.48±0.11 O.D.; n=6). In contrast, endothelium-dependent relaxations to β-agonist isoproterenol were significantly enhanced in APP –/– mice aortas (P<0.05; n=10) while endothelium-dependent relaxations to acetylcholine were unaltered (P=n.s.; n=12). Incubation of aortic rings with indomethacin significantly impaired relaxations to isoproterenol as well as acetylcholine in APP –/– mice (P<0.05; n=8) while concomitant treatment with NOS inhibitor L-NAME completely abolished relaxations to both agonists (P<0.05; n=6-7). Incubation of aortic rings with isoproterenol significantly increased cAMP in the aortas of APP –/– mice (16.2±4.1 pmol/mg; P<0.05 vs. WT: 6.6±4.1 pmol/mg; n=7). Furthermore, cAMP levels were significantly enhanced by acetylcholine in APP –/– mice aortas (38±9 pmol/mg; P<0.05 vs. WT: 14±3 pmol/mg; n=8) while acetylcholine stimulated cGMP levels were reduced (59±5 pmol/mg; P<0.05 vs. WT: 83±7 pmol/mg; n=12). Our results suggest that increased circulating levels of catecholamines in APP –/– mice are responsible for observed vascular phenotype. These findings indicate that under physiological conditions, APP expression plays an important role in control of vascular endothelial function .

There is no therapy that promotes maturation and functionality of a dialysis arteriovenous fistula (AVF). The search for such therapies largely relies on evaluation of vascular responses and putative therapies in experimental AVFs. We studied an AVF in mice with chronic kidney disease (CKD). We demonstrate numerous stressors in the vein of the AVF-CKD group, including pathological shear, mitogenic, inflammatory, and hypoxia-reoxygenation stress. Because stress promotes premature senescence, we examined whether senescence is induced in the vein of the AVF-CKD model. We demonstrate a senescence phenotype in the AVF-CKD model, as indicated by increased expression of p16Ink4a, p21Cip1, and p53 and expected changes for certain senescence-associated microRNAs. RNA-sequencing analysis demonstrated differential expression of ~10,000 genes, including upregulation of proinflammatory and proliferative genes, in the vein of the AVF-CKD group. The vein in the AVF-CKD group exhibited telomere erosion and increased senescence-associated β-galactosidase activity and staining. Senescence was induced in the artery of the AVF-CKD group and in the vein of the AVF without CKD. Finally, given the rapidly rising clinical interest in senolytics, we provide proof of concept of senolytics as a therapeutic approach by demonstrating that senolytics decrease p16Ink4a expression in the AVF-CKD model. This study introduces a novel concept underlying the basis for maturational and functional failure in human dialysis AVFs and identifies a new target for senolytic therapy.