CXCL1 presented on the luminal endothelial surface leads to an increase in the number of peri-collateral macrophages, thus improving the arteriogenic response after arterial ligation.

Background-Angiogenesis is a natural mechanism to restore perfusion to the ischemic myocardium after acute myocardial infarction (MI). Therapeutic angiogenesis is being explored as a novel treatment for MI patients; however, sensitive, noninvasive in vivo measures of therapeutic efficacy are lacking and need to be developed. Here, a molecular magnetic resonance imaging method is presented to noninvasively image angiogenic activity in vivo in a murine model of MI with cyclic Asn-Gly-Arg (cNGR)-labeled paramagnetic quantum dots (pQDs). The tripeptide cNGR homes specifically to CD13, an aminopeptidase that is strongly upregulated during myocardial angiogenesis. Methods and Results-Acute MI was induced in male Swiss mice via permanent ligation of the left anterior descending coronary artery. Molecular magnetic resonance imaging was performed 7 days after surgery and up to 2 hours after intravenous contrast agent administration. Injection of cNGR-pQDs resulted in a strong negative contrast that was located mainly in the infarcted myocardium. This negative contrast was significantly less in MI mice injected with unlabeled pQDs and in sham-operated mice injected with cNGR-pQDs. Validation with ex vivo 2-photon laser scanning microscopy revealed a strong colocalization of cNGR-pQDs with vascular endothelial cells, whereas unlabeled pQDs were mostly extravasated and diffused through the tissue. Additionally, 2-photon laser scanning microscopy demonstrated significant microvascular remodeling in the infarct/border zones compared with remote myocardium. Conclusions-cNGR-pQDs allow selective, noninvasive detection of angiogenic activity in the infarcted heart with the use of in vivo molecular magnetic resonance imaging and ex vivo 2-photon laser scanning microscopy. (Circulation. 2010; 121:775-783.)

The bone morphogenetic protein receptor II (BMPRII) signaling pathway is impaired in pulmonary arterial hypertension and mutations in the BMPR2 gene have been observed in both heritable and idiopathic pulmonary arterial hypertension. However, all BMPR2 mutation carriers do not develop pulmonary arterial hypertension, and inflammation could trigger the development of the disease in BMPR2 mutation carriers. Circulating levels and/or lung tissue expression of cytokines such as tumor necrosis factor-α or interleukin-18 are elevated in patients with pulmonary arterial hypertension and could be involved in the pathogenesis of pulmonary arterial hypertension. We consequently hypothesized that cytokines could trigger endothelial dysfunction in addition to impaired BMPRII signaling. Our aim was to determine whether impairment of BMPRII signaling might affect endothelium barrier function and adhesiveness to monocytes, in response to cytokines. BMPR2 was silenced in human lung microvascular endothelial cells (HLMVECs) using lentiviral vectors encoding microRNA-based hairpins. Effects of tumor necrosis factor-α and interleukin-18 on HLMVEC adhesiveness to the human monocyte cell line THP-1, adhesion molecule expression, endothelial barrier function and activation of P38MAPK were investigated in vitro. Stable BMPR2 silencing in HLMVECs resulted in impaired endothelial barrier function and constitutive activation of P38MAPK. Adhesiveness of BMPR2-silenced HLMVECs to THP-1 cells was enhanced by tumor necrosis factor-α and interleukin-18 through ICAM-1 adhesion molecule. Interestingly, tumor necrosis factor-α induced activation of P38MAPK and disrupted endothelial barrier function in BMPR2-silenced HLMVECs. Altogether, our findings showed that stable BMPR2 silencing resulted in impaired endothelial barrier function and activation of P38MAPK in HLMVECs. In BMPR2-silenced HLMVECs, cytokines enhanced adhesiveness capacities, activation of P38MAPK and impaired endothelial barrier function suggesting that cytokines could trigger the development of pulmonary arterial hypertension in a context of impaired BMPRII signaling pathway.

During angiogenesis, endothelial tip cells start sprouting and express delta-like 4 (DLL4) downstream of vascular endothelial growth factor (VEGF). DLL4 subsequently activates Notch in the adjacent stalk cells suppressing sprouting. VEGF also activates A disintegrin and metalloproteases (ADAMs) that induce Notch ectodomain shedding. Although two major ADAMs, i.e. ADAM10 and ADAM17, have been implicated in Notch-signalling activation, their apparent different roles in angiogenesis have not been fully understood yet. The objective of this study was to determine the roles of ADAM10 and ADAM17 activity in angiogenesis. In mouse retinas, ADAM10 or γ-secretase inhibition induced vascular sprouting and density in vivo, whereas attenuation of both ADAM10 and ADAM17 activity produced the opposite phenotype. Retinal blood vessel analysis in ADAM17 hypomorphic mice confirmed the requirement for ADAM17 activity in angiogenesis. However, ADAM17 inhibition did not phenocopy blood vessel increase by Notch blockage. These observations suggest that ADAM17 regulates other fundamental players during angiogenesis besides Notch, which were not affected by ADAM10. By means of an angiogenesis proteome assay, we found that ADAM17 inhibition induced the expression of a naturally occurring inhibitor of angiogenesis Thrombospondin 1 (TSP1), whereas ADAM10 inhibition did not. Accordingly, ADAM17 overexpression downregulated TSP1 expression, and the TSP1 inhibitor LSKL rescued angiogenesis in the tube formation assay downstream of VEGF in the presence of ADAM17 inhibition. Finally, genetic and pharmacological ADAM17 blockade resulted in increased TSP1 expression in mouse retina. Altogether, our results show that ADAM10 and ADAM17 have opposite effects on sprouting angiogenesis that may be unrelated to Notch signalling and involves differentially expressed anti-angiogenic proteins such as TSP1.

AimsEnhancement of collateral development in coronary or peripheral artery disease is a therapeutic target, but it has proven difficult to achieve. Macrophages are key players in collateral remodeling, yet the effect of different macrophage subsets on arteriogenesis has not been investigated.Methods and ResultsMurine macrophages were cultured from bone marrow and polarized into M1 (IFNγ), M2a (IL-4) or M2c (IL-10) subsets. C57BL/6 mice underwent femoral artery ligation followed by intramuscular injection of macrophage subsets. Using eGFP expressing macrophages, cells could be detected at least 6 days after ligation and were located in the perivascular space of collateral vessels. After 14 days, perfusion ratio was increased in animals treated with M1 as well as M2a and M2c macrophages compared to control. Depletion of circulating monocytes by clodronate liposome injections did not hamper reperfusion recovery, however, treatment with exogenous polarized macrophages improved perfusion ratio after 14 days again. We used IL10Rfl/fl/LysMCre+ mice to study the effect of inhibition of endogenous polarization towards specifically M2c macrophages on arteriogenesis. Deletion of the IL10-receptor (IL10R) in the myeloid lineage did not affect reperfusion recovery, yet the pro-arteriogenic effect of exogenously injected M2c macrophages was still present.ConclusionsLocal injection of polarized macrophages promotes reperfusion recovery after femoral artery ligation and is not influenced by depletion of circulatory monocytes. Preventing endogenous M2c polarization did not affect reperfusion recovery suggesting that M2c’s are not required for collateralization, but are sufficient to induce collateral formation upon exogenous administration. This is the first study using local injection of macrophage subsets showing the pro-arteriogenic effect of polarized macrophages.

Objective— Notch has been implicated in neointima formation as reflected by increased Notch/Jagged expression on vascular injury and the promigratory effect of Notch signaling on smooth muscle cells. Soluble Jagged-1 (sJag1) has been shown to inhibit Notch signaling in vitro; however, its capacity to suppress neointima formation remains unknown. Methods and Results— Balloon injury of rat carotid arteries induced Notch1, Notch3, and Jagged-1 expression at days 3 and 14 postinjury. Notch signaling was activated as shown by increased expression of the Notch target gene Herp2. Adenoviral sJag1 (Ad-sJag1) transfection reduced neointima formation in carotid artery and enhanced reendothelialization, whereas adenoviral full-length Jagged-1 (Ad-Fl-Jag1) or LacZ had no effect. Injury-induced Herp2 expression was absent in vessels treated with Ad-sJag1. Consistently, Herp2 expression was reduced in Ad-sJag1-infected or recombinant sJag1 –treated coronary artery smooth muscle cells (CASMCs). Ad-sJag1 had no effect on human umbilical endothelial cell behavior, but it significantly reduced proliferation and migration of CASMCs. Overexpression of Herp2 in sJag1-treated CASMCs rescued the migratory and proliferative capacity in vitro. Conclusion— Our results demonstrate that sJag1 can inhibit neointima formation after balloon injury by decreasing smooth muscle cell proliferation and migration through interference with Notch-Herp2 signaling.

BackgroundAutologous arteriovenous (AV) fistulas are the first choice for vascular access but have a high risk of non-maturation due to insufficient vessel adaptation, a process dependent on nitric oxide (NO)-signaling. Chronic kidney disease (CKD) is associated with oxidative stress that can disturb NO-signaling. Here, we evaluated the influence of CKD on AV fistula maturation and NO-signaling.MethodsCKD was established in rats by a 5/6th nephrectomy and after 6 weeks, an AV fistula was created between the carotid artery and jugular vein, which was followed up at 3 weeks with ultrasound and flow assessments. Vessel wall histology was assessed afterwards and vasoreactivity of carotid arteries was studied in a wire myograph. The soluble guanylate cyclase (sGC) activator BAY 60–2770 was administered daily to CKD animals for 3 weeks to enhance fistula maturation.ResultsCKD animals showed lower flow rates, smaller fistula diameters and increased oxidative stress levels in the vessel wall. Endothelium-dependent relaxation was comparable but vasorelaxation after sodium nitroprusside was diminished in CKD vessels, indicating NO resistance of the NO-receptor sGC. This was confirmed by stimulation with BAY 60–2770 resulting in increased vasorelaxation in CKD vessels. Oral administration of BAY 60–2770 to CKD animals induced larger fistula diameters, however; flow was not significantly different from vehicle-treated CKD animals.ConclusionsCKD induces oxidative stress resulting in NO resistance that can hamper AV fistula maturation. sGC activators like BAY 60–2770 could offer therapeutic potential to increase AV fistula maturation.

The separate and combined GH-lowering effects of single doses of octreotide and bromocriptine were assessed in 51 acromegalic patients on 4 occasions each 2 days apart. Patients received sequentially: placebo sc (N=51), 50 \ g=m\ g octreotide sc (N=51), 2.5 mg bromocriptine po (N=40) or a combination of both drugs (N=25). With octreotide, in 28 patients (55%) GH levels were suppressed to less than 5 \g=m\g/land 39 of them (76.5%) had a 50% or greater decrease of their basal GH level from 2 to 6 h. During bromocriptine, GH values were suppressed to below 5 \g=m\g/l in 11 patients (27.5%) and reduced by 50% or more in 21 (52.5%). The combination of both drugs acutely suppressed GH levels to less than 2 \g=m\g/l in 32%, to less than 5 \g=m\g/l in 56%, and by more than 50% in 84% of patients. Octreotide produced a stronger and faster suppression of GH levels than bromocriptine in most patients. The combination of both drugs had an additive effect on the lowering of GH levels, especially between 7 and 10 h after drug administration. These results suggest that chronic therapy with a combination of both drugs may be as effective as therapy with higher doses of either compound alone. Albeit transient, octreotide caused a rapid near total suppression of insulin release in the morning, 15 to 45 min after administration. Postprandial glucose rise, between 2 and 3.5 h after breakfast was significantly higher during octreotide than on the control day.Medical therapy of acromegaly has been greatly improved in recent years following the availability of the long-acting somatostatin analogue, octreo¬ tide (Sandostatin®, SMS 201-995). Comparative studies between the acute inhibitory effects of bro¬ mocriptine (Parlodel®) and those of octreotide, on GH secretion in small numbers of acromegalic pa¬ tients indicate that the majority of these patients show a significantly higher sensitivity to octreotide than to bromocriptine (1-3).Chronic therapy with octreotide produces rapid clinical improvement in virtually all acromegalic patients, which is accompanied by a decrease in circulating GH and IGF-I levels. However, com¬ plete normalization of mean 24-h GH and IGF-I levels is reached in only approximately 50% of pa¬ tients during intermittent sc administration of 50-100 pg octreotide twice or thrice daily (2,(4)(5)(6)(7)(8)(9)(10)(11). It has been hypothesized that the absence of complete suppression of GH levels throughout the 24-h period is related to the pharmacokinetics of octreo¬ tide, which allows an increase in tumoural hor¬ mone secretion during the hours immediately preceeding the next injection (5)(6)(7)(8)(9)12). Indeed, in stud¬ ies in which octreotide was administered by 24 h sc infusion, acromegalic patients tended to show better control of GH secretion over the 24-h period, with concomitant normalization of circulat¬ ing IGF-I levels occurring more frequently (9,(11)(12)(13)).In the present study we have investigated whether octreotide and bromocriptine given in combination to acromegalic patients might result in a more ...