Angiotensin receptor-neprilysin inhibitor improves coronary collateral perfusion

BACKGROUND: We investigated the pleiotropic effects of an angiotensin receptor-neprilysin inhibitor (ARNi) on collateral-dependent myocardial perfusion in a rat model of coronary arteriogenesis, and performed comprehensive analyses to uncover the underlying molecular mechanisms. METHODS: A rat model of coronary arteriogenesis was established by implanting an inflatable occluder on the left anterior descending coronary artery followed by a 7-day repetitive occlusion procedure (ROP). Coronary collateral perfusion was measured by using a myocardial particle infusion technique. The putative ARNi-induced pro-arteriogenic effects were further investigated and compared with an angiotensin-converting enzyme inhibitor (ACEi). Expression of the membrane receptors and key enzymes in the natriuretic peptide system (NPS), renin-angiotensin-aldosterone system (RAAS) and kallikrein-kinin system (KKS) were analyzed by quantitative polymerase chain reaction (qPCR) and immunoblot assay, respectively. Protein levels of pro-arteriogenic cytokines were measured by enzyme-linked immunosorbent assay, and mitochondrial DNA copy number was assessed by qPCR due to their roles in arteriogenesis. Furthermore, murine heart endothelial cells (MHEC5-T) were treated with a neprilysin inhibitor (NEPi) alone, or in combination with bradykinin receptor antagonists. MHEC5-T proliferation was analyzed by colorimetric assay. RESULTS: The in vivo study showed that ARNis markedly improved coronary collateral perfusion, regulated the gene expression of KKS, and increased the concentrations of relevant pro-arteriogenic cytokines. The in vitro study demonstrated that NEPis significantly promoted MHEC5-T proliferation, which was diminished by bradykinin receptor antagonists. CONCLUSION: ARNis improve coronary collateral perfusion and exert pro-arteriogenic effects via the bradykinin receptor signaling pathway

In situ proliferation and differentiation of macrophages in dental pulp

The presence of macrophages in dental pulp is well known. However, whether these macrophages proliferate and differentiate in the dental pulp in situ, or whether they constantly migrate from the blood stream into the dental pulp remains unknown. We have examined and compared the development of dental pulp macrophages in an organ culture system with in vivo tooth organs to clarify the developmental mechanism of these macrophages. The first mandibular molar tooth organs from ICR mice aged between 16 days of gestation (E16) to 5 days postnatally were used for in vivo experiments. Those from E16 were cultured for up to 14 days with or without 10% fetal bovine serum. Dental pulp tissues were analyzed with immunohistochemistry to detect the macrophages and with reverse transcription and the polymerase chain reaction (RT-PCR) for the detection of factors related to macrophage development. The growth curves for the in vivo and in vitro cultured cells revealed similar numbers of F4/80-positive macrophages in the dental pulp. RT-PCR analysis indicated the constant expression of myeloid colony-stimulating factor (M-CSF) in both in-vivo- and in-vitro-cultured dental pulp tissues. Anti-M-CSF antibodies significantly inhibited the increase in the number of macrophages in the dental pulp. These results suggest that (1) most of the dental pulp macrophages proliferate and differentiate in the dental pulp without a supply of precursor cells from the blood stream, (2) M-CSF might be a candidate molecule for dental pulp macrophage development, and (3) serum factors might not directly affect the development of macrophages

Adaptation of external counterpulsation based on individual shear rate therapy improves endothelial function and claudication distance in peripheral artery disease

BACKGROUND: External counterpulsation therapy enhances blood flow and was shown to improve endothelial function and quality of life in coronary artery disease patients. However, high pressures of up to 300 mmHg may lead to malperfusion of the ischaemic limb. To improve the clinical outcome of patients with peripheral artery disease (PAD), we adjusted external counterpulsation and developed a novel non-invasive approach termed individual shear rate therapy (ISRT). PATIENTS AND METHODS: In the present study, 14 patients with a Fontaine stage IIb and femoral-popliteal PAD underwent 30 hours of ISRT over 5 weeks. For ISRT, individual treatment pressures that do not exceed 160 mmHg were assessed by Doppler flow parameters during counterpulsation (individual shear rate diagnosis) in order to enhance and maximise peripheral perfusion. The study aimed to enhance peripheral perfusion and evaluate the primary clinical endpoint endothelial function, as well as to perform preliminary analysis of the ankle brachial index (ABI) and walking distance. RESULTS: Doppler flow measurements in the lower limb (ankle) validated that maximum blood flow velocity during systole and acceleration doubled during ISRT. Study results demonstrated that long-term ISRT significantly increased flow-mediated dilation (FMD) in the brachial artery (0.13+/- 0.09 mm to 0.38+/- 0.05 mm; p < 0.05), while nitromediated dilation (0.36+/- 0.10 mm to 0.45+/- 0.08 mm) remained and common femoral artery FMD did not reach statistical significance (0.38+/- 0.08 mm to 0.67+/- 0.19 mm; p<0.05). Initial claudication distance considerably improved for all patients after 30 hours of ISRT (92.6 +/- 8.2 metres to 280+/- 101.3 metres, p<0.05), just like the absolute claudication distance, which showed a more than 2.5-fold increase (167.8+/- 18.1 metres to 446.7+/- 133.3 metres; p<0.05). The ABI did not improve (0.58+/- 0.03 to 0.65+/- 0.04). CONCLUSIONS: Our data demonstrate that long-term ISRT is a potential novel non-invasive treatment to improve endothelial function and absolute pain-free walking distance for PAD patients

Granulocyte colony-stimulating factor improves cerebrovascular reserve capacity by enhancing collateral growth in the circle of Willis

BACKGROUND AND PURPOSE: Restoration of cerebrovascular reserve capacity (CVRC) depends on the recruitment and positive outward remodeling of preexistent collaterals (arteriogenesis). With this study, we provide functional evidence that granulocyte colony-stimulating factor (G-CSF) augments therapeutic arteriogenesis in two animal models of cerebral hypoperfusion. We identified an effective dosing regimen that improved CVRC and stimulated collateral growth, thereby improving the outcome after experimentally induced stroke. METHODS: We used two established animal models of (a) cerebral hypoperfusion (mouse, common carotid artery ligation) and (b) cerebral arteriogenesis (rat, 3-vessel occlusion). Following therapeutic dose determination, both models received either G-CSF, 40 mug/kg every other day, or vehicle for 1 week. Collateral vessel diameters were measured following latex angiography. Cerebrovascular reserve capacities were assessed after acetazolamide stimulation. Mice with left common carotid artery occlusion (CCAO) were additionally subjected to middle cerebral artery occlusion, and stroke volumes were assessed after triphenyltetrazolium chloride staining. Given the vital role of monocytes in arteriogenesis, we assessed (a) the influence of G-CSF on monocyte migration in vitro and (b) monocyte counts in the adventitial tissues of the growing collaterals in vivo. RESULTS: CVRC was impaired in both animal models 1 week after induction of hypoperfusion. While G-CSF, 40 mug/kg every other day, significantly augmented cerebral arteriogenesis in the rat model, 50 or 150 mug/kg every day did not show any noticeable therapeutic impact. G-CSF restored CVRC in mice (5 +/- 2 to 12 +/- 6%) and rats (3 +/- 4 to 19 +/- 12%). Vessel diameters changed accordingly: in rats, the diameters of posterior cerebral arteries (ipsilateral: 209 +/- 7-271 +/- 57 mum; contralateral: 208 +/- 11-252 +/- 28 mum) and in mice the diameter of anterior cerebral arteries (185 +/- 15-222 +/- 12 mum) significantly increased in the G-CSF groups compared to controls. Stroke volume in mice (10 +/- 2%) was diminished following CCAO (7 +/- 4%) and G-CSF treatment (4 +/- 2%). G-CSF significantly increased monocyte migration in vitro and perivascular monocyte numbers in vivo. CONCLUSION: G-CSF augments cerebral collateral artery growth, increases CVRC and protects from experimentally induced ischemic stroke. When comparing three different dosing regimens, a relatively low dosage of G-CSF was most effective, indicating that the common side effects of this cytokine might be significantly reduced or possibly even avoided in this indication

Erratum: Granulocyte Colony-Stimulating Factor Improves Cerebrovascular Reserve Capacity by Enhancing Collateral Growth in the Circle of Willis

<i>Background and Purpose:</i> Restoration of cerebrovascular reserve capacity (CVRC) depends on the recruitment and positive outward remodeling of preexistent collaterals (arteriogenesis). With this study, we provide functional evidence that granulocyte colony-stimulating factor (G-CSF) augments therapeutic arteriogenesis in two animal models of cerebral hypoperfusion. We identified an effective dosing regimen that improved CVRC and stimulated collateral growth, thereby improving the outcome after experimentally induced stroke. <i>Methods:</i> We used two established animal models of (a) cerebral hypoperfusion (mouse, common carotid artery ligation) and (b) cerebral arteriogenesis (rat, 3-vessel occlusion). Following therapeutic dose determination, both models received either G-CSF, 40 µg/kg every other day, or vehicle for 1 week. Collateral vessel diameters were measured following latex angiography. Cerebrovascular reserve capacities were assessed after acetazolamide stimulation. Mice with left common carotid artery occlusion (CCAO) were additionally subjected to middle cerebral artery occlusion, and stroke volumes were assessed after triphenyltetrazolium chloride staining. Given the vital role of monocytes in arteriogenesis, we assessed (a) the influence of G-CSF on monocyte migration in vitro and (b) monocyte counts in the adventitial tissues of the growing collaterals in vivo. <i>Results:</i> CVRC was impaired in both animal models 1 week after induction of hypoperfusion. While G-CSF, 40 µg/kg every other day, significantly augmented cerebral arteriogenesis in the rat model, 50 or 150 µg/kg every day did not show any noticeable therapeutic impact. G-CSF restored CVRC in mice (5 ± 2 to 12 ± 6%) and rats (3 ± 4 to 19 ± 12%). Vessel diameters changed accordingly: in rats, the diameters of posterior cerebral arteries (ipsilateral: 209 ± 7–271 ± 57 µm; contralateral: 208 ± 11–252 ± 28 µm) and in mice the diameter of anterior cerebral arteries (185 ± 15–222 ± 12 µm) significantly increased in the G-CSF groups compared to controls. Stroke volume in mice (10 ± 2%) was diminished following CCAO (7 ± 4%) and G-CSF treatment (4 ± 2%). G-CSF significantly increased monocyte migration in vitro and perivascular monocyte numbers in vivo. <i>Conclusion:</i> G-CSF augments cerebral collateral artery growth, increases CVRC and protects from experimentally induced ischemic stroke. When comparing three different dosing regimens, a relatively low dosage of G-CSF was most effective, indicating that the common side effects of this cytokine might be significantly reduced or possibly even avoided in this indication

Arteriogenesis is modulated on bradykinin receptor signaling

Background: Positive outward remodeling of preexisting collateral arteries into functional conductance arteries, arteriogenesis, is a major endogenous rescue mechanism to prevent for cardiovascular ischemia. Collateral arterial growth is accompanied by kininogen expression in growing arteries, the precursor of kinins, which signal via the kinin receptors 1 (B1R) and kinin receptor 2 (B2R). The purpose of this study was to elucidate the functional role and mechanism of bradykinin receptor signaling in arteriogenesis. Methods and Results: Bradykinin receptors positively affected arteriogenesis, with the contribution of B1R being more pronounced than B2R. In mice, arteriogenesis on femoral artery occlusion was significantly reduced in B1R mutant mice as evidenced by reduced microspheres and laser Doppler flow perfusion measurements. Transplantation of wild-type bone marrow cells into irradiated B1R mutant mice restored arteriogenesis, whereas bone marrow chimeric mice generated by reconstituting wild-type mice with B1R mutant bone marrow showed reduced arteriogenesis after femoral artery occlusion. In the rat brain 3-vessel occlusion arteriogenesis model, pharmacological treatment of B1R inhibited arteriogenesis and stimulation of B1R enhanced arteriogenesis. In the rat, femoral artery ligation combined with arterial venous shunt model resulted in flow-driven arteriogenesis, and treatment with B1R antagonist R715 decreased vascular remodeling and leukocyte invasion (monocytes) into the perivascular tissue. In monocyte migration assays, in vitro B1R agonists enhanced migration of monocytes. Conclusions: Kinin receptors act as positive modulators of arteriogenesis in mice and rats. B1R can be blocked or therapeutically stimulated by B1R antagonists or agonists, respectively, involving a contribution of peripheral immune cells (monocytes) linking hemodynamic conditions with inflammatory pathways