Citations

Tetrahydrobiopterin (BH 4 ) is an essential cofactor for endothelial nitric oxide synthase (eNOS). Suboptimal level of BH 4 reduces formation of nitric oxide leading to uncoupling of eNOS. However, the exact molecular mechanisms underlying the effects of eNOS uncoupling on vascular endothelium in‐vivo are not completely understood. In the present study, we used the hph‐1 mouse, which displays GTP‐cyclohydrolase I deficiency. HPLC analysis revealed that BH 4 levels were significantly reduced in the aortas of hph‐1 mice (3.9±0.5 pmol/mg; P<0.05 vs. wild‐type mice: 5.9±0.6 pmol/mg), while oxidative products of BH 4 , 7,8‐dihydrobiopterin (7,8‐BH 2 ), were unchanged. Consequently, the BH 4 to 7,8‐BH 2 ratio was significantly reduced in the aorta of hph‐1 mice (P<0.05). Expression of eNOS protein was not different between wild‐type and hph‐1 mice. Unexpectedly, protein expressions of phosphorylated Ser 1177 ‐eNOS and Ser 473 ‐Akt1 were significantly increased in hph‐1 mice aortas (P<0.05). Consistent with uncoupling of eNOS, L‐NAME sensitive superoxide anion production and 3‐nitrotyrosine formation were significantly increased in aortas of hph‐1 mice (P<0.05). Protein expressions of CuZnSOD, MnSOD, and EC‐SOD were unaltered in the aortas of hph‐1 mice as compared with wild‐type mice. Our results suggest that uncoupling eNOS increases phosphorylation of Akt1 and eNOS. This effect is most likely mediated by increased formation of eNOS‐derived reactive oxygen species including superoxide anion and hydrogen peroxide.

Impaired endothelial function in cardiovascular disorders is mediated, in part, by uncoupling of eNOS caused by reduced availability of tetrahydrobiopterin (BH 4 ). Endothelial progenitor cells (EPCs), originating from bone marrow, contribute to repair of vascular system by either neovascularization following tissue ischemia or re‐endothelialization after endothelial injury. The contribution of BH 4 to the angiogenic function has not been studied. In the present study, we investigated the role of BH 4 in angiogenesis and mobilization of cells enriched in EPCs in hph‐1 mutant mice, a genetic model of GTP‐cyclohydrolase I deficiency. HPLC analysis revealed that enzymatic activity of GTP‐cyclohydrolase I is reduced by 65% in bone marrow of hph‐1 mice (22±4 pmol neopterin/mg/h; P<0.05 vs. wild‐type mice: 63±4 pmol/mg/h). Basal counts of cells co‐expressing CD34 and VEGFR2 (Flk‐1) were reduced in bone marrow and peripheral blood of hph‐1 mice. Fourteen days after hind limb ischemia, recovery of blood flow was significantly reduced in hph‐1 mice as compared to blood flow in ischemic limb of wild type mice. Our findings suggest that reduced bioavailability of BH 4 impairs mobilization of cells enriched in EPCs. This effect may help to explain impaired angiogenesis in hph‐1 mice.

High salt diet (HS) impairment of endothelial nitric oxide (NO) signaling has been described in human and rodents. The mechanisms which mediate this dysfunction remain unknown. We and others have previously shown histone deacetylase 1 (HDAC1) decreases endothelial NO production. We therefore sought to investigate the mechanisms by which HS initiates endothelial dysfunction and if it is HDAC1‐dependent. We first investigated if HS induced endothelial dysfunction is acutely regulated by a circulating factor. Male Sprague Dawley rats were fed normal salt diet (NS; 0.49% NaCl) or 2 weeks of HS (4.0% NaCl). Afferent arteriole vasoconstrictor responses to the NOS inhibitor, L‐NAME, were monitored using the in vitro, blood perfused juxtamedullary nephron preparation. Arterioles from HS fed rats perfused with blood from HS fed rats demonstrated blunted constriction to L‐NAME (96 ± 2% of Con) compared to arterioles from NS fed rats perfused with blood from NS fed rats (76.2 ± 2% of Con; P=0.0017). Perfusing arterioles from NS fed rats with blood HS fed rats blunted constriction to L‐NAME (88 ± 1% of Con) compared to perfusing with blood from NS fed rats (P=0.0003), but not to the same extent as arterioles from HS fed rats perfused with blood from HS fed rats (P=0.0095). HS arterioles perfused with blood from NS fed rats had similar L‐NAME‐induced constriction (75 ± 2% of Con) compared to arterioles from NS fed rats perfused with blood from NS fed rats (P=0.9107). In short, the impaired constriction to L‐NAME is dependent up on the diet of the blood donor and not the diet of the arteriole donor suggesting HS impairs endothelial function through an acute‐acting circulating factor. This was not due to increased plasma arginase activity (NS: 7.96 ± 3.73 U/L vs HS: 5.42 ± 1.83 U/L P=0.5813), changes in plasma tetrahydrobiopterin (NS: 65.09 ± 11.57 pmol/ml vs HS: 53.54 ± 5.59 pmol/ml, P=0.311) or changes in plasma dihydrobiopterin (NS: 98.18 ± 7.84 pmol/ml vs HS: 87.45 ± 13.28 pmol/ml, P=0.530). We then investigated if HS increased endothelial HDAC1 activity. Renal endothelial cells were isolated from NS and HS fed rats and incubated in plasma from the same rat. HDAC1 activity was defined as the MS‐275 (HDAC1 specific inhibitor) inhibitable portion of total HDAC activity. We found HS significantly increased renal endothelial HDAC1 activity (NS: 0.38 ± 0.03 pmol/min vs HS:0.94 ± 0.19 pmol/min, P=0.01). Furthermore, in vitro HDAC1 inhibition with 300 nM MS‐275 restored the arteriolar vasoconstrictor responses in HS rats (79 ± 3% of Con) to values similar to NS fed rats (P=0.969) and to NS rats + MS‐275 (79 ± 1% of Con; P>0.998). When plasma from HS fed rats was combined with erythrocytes from NS fed rats and perfused through arterioles from NS fed rats, constriction to L‐NAME was blunted (85 ± 3% of Con; P=0.0129). Under these same conditions, HDAC1 inhibition restored constriction (75.7 ± 0.95 % of Con) similar to controls (P=0.979). We conclude that HS disruption of renal microvascular NO signaling is initiated by a circulating factor(s) that increases endothelial HDAC1 activity.

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.

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.

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 .

The mechanisms underlying dysfunction of cerebral microvasculature induced by type 1 diabetes (T1D) are not fully understood. We hypothesized that in cerebral microvascular endothelium, α-processing of amyloid precursor protein (APP) is impaired by T1D. In cerebral microvessels derived from streptozotocin (STZ)-induced T1D mice protein levels of APP and its α-processing enzyme, a disintegrin and metalloprotease 10 (ADAM10) were significantly decreased, along with down-regulation of adenylate cyclase 3 (AC3) and enhanced production of thromboxane A 2 (TXA 2 ). In vitro studies in human brain microvascular endothelial cells (BMECs) revealed that knockdown of AC3 significantly suppressed ADAM10 protein levels, and that activation of TXA 2 receptor decreased APP expression. Furthermore, levels of soluble APPα (sAPPα, a product of α-processing of APP) were significantly reduced in hippocampus of T1D mice. In contrast, amyloidogenic processing of APP was not affected by T1D in both cerebral microvessels and hippocampus. Most notably, studies in endothelial specific APP knockout mice established that genetic inactivation of APP in endothelium was sufficient to significantly reduce sAPPα levels in the hippocampus. In aggregate, our findings suggest that T1D impairs non-amyloidogenic processing of APP in cerebral microvessels. This may exert detrimental effect on local concentration of neuroprotective molecule, sAPPα, in the hippocampus.

The exact physiological function of amyloid-β precursor protein (APP) in endothelial cells is unknown. Endothelium-specific APP-deficient (eAPP −/− ) mice were created to gain new insights into the role of APP in the control of vascular endothelial function. Endothelium-dependent relaxations to acetylcholine were significantly impaired in basilar arteries of global APP knockout (APP −/− ) and eAPP −/− mice ( P < 0.05). In contrast, endothelium-independent relaxations to nitric oxide (NO)-donor diethylamine-NONOate were unchanged. Western blot analysis revealed that protein expression of endothelial nitric oxide synthase (eNOS) was significantly downregulated in large cerebral arteries of APP −/− mice and eAPP −/− mice as compared to respective wild-type littermates ( P < 0.05). Furthermore, basal levels of cyclic guanosine monophosphate (cGMP) were also significantly reduced in large cerebral arteries of APP-deficient mice ( P < 0.05). In contrast, protein expression of prostacyclin synthase as well as levels of cyclic adenosine monophosphate (cAMP) was not affected by genetic inactivation of APP in endothelial cells. By using siRNA to knockdown APP in cultured human brain microvascular endothelial cells we also found a significant downregulation of eNOS mRNA and protein expressions in APP-deficient endothelium ( P < 0.05). These findings indicate that under physiological conditions, expression of APP in cerebral vascular endothelium plays an important protective function by maintaining constitutive expression of eNOS .

Amyloid precursor protein (APP) is evolutionary conserved protein expressed in endothelial cells of cerebral and peripheral arteries. In this review, we discuss mechanisms responsible for expression and proteolytic cleavage of APP in endothelial cells. We focus on physiological and pathological implications of APP expression in vascular endothelium.