Pulmonary arterial hypertension drugs can partially restore altered angiogenic capacities in bmpr2-silenced human lung microvascular endothelial cells.

Mutations in the bone morphogenetic protein receptor type 2 (bmpr2) gene and signaling pathway impairment are observed in heritable and idiopathic pulmonary arterial hypertension (PAH). In PAH, endothelial dysfunction is currently handled by drugs targeting the endothelin-1 (ET-1), nitric oxide (NO), and prostacyclin (PGI2) pathways. The role of angiogenesis in the disease process and the effect of PAH therapies on dysregulated angiogenesis remain inconclusive. We aim to investigate in vitro whether (i) bmpr2 silencing can impair angiogenic capacity of human lung microvascular endothelial cells (HLMVECs) and (ii) PAH therapies can restore them. The effects of macitentan (ET-1), tadalafil (NO), and selexipag (PGI2), on BMPRII pathway activation, endothelial barrier function, and angiogenesis were investigated in bmpr2-silenced HLMVECs. Stable bmpr2 silencing resulted in impaired migration and tube formation in vitro capacity. Inhibition of ET-1 pathway was able to partially restore tube formation in bmpr2-silenced HLMVECs, whereas none of the therapies was able to restore endothelial barrier function, no deleterious effects were observed. Our findings highlight the potential role of BMPRII signaling pathway in driving pulmonary endothelial cell angiogenesis. In addition, PAH drugs display limited effects on endothelial function when BMPRII is impaired, suggesting that innovative therapeutic strategies targeting BMPRII signaling are needed to better rescue endothelial dysfunction in PAH.

Impairment of Angiogenesis-Driven Clot Resolution Is a Key Event in the Progression to Chronic Thromboembolic Pulmonary Hypertension: Validation in a Novel Rabbit Model.

BACKGROUND: Chronic thromboembolic pulmonary hypertension (CTEPH) is a life-threatening condition and rare complication of acute pulmonary embolism. Mechanisms underlying impaired clot resolution and in sustained fibrothrombotic obstruction of the pulmonary arterial bed remain poorly understood. Since defective angiogenesis correlated to defective clot resolution based on observations in surgical material from patients with CTEPH, we aimed to validate its crucial pathogenic role by intrathrombus inhibition of angiogenesis in a novel CTEPH rabbit model. METHODS: We aimed to compare whether intrathrombus administration of an antifibrinolytic agent, tranexamic acid, or an inhibitor of angiogenesis, SU5416, would contribute to CTEPH progression. Both products were administered on a weekly basis by autologous clot embolization in rabbits. Right ventricular pressure was monitored by telemetry, right ventricular function by transthoracic echocardiography, and a complete pulmonary hemodynamic evaluation was obtained through right heart catheterization. Markers of inflammation, endothelial dysfunction, heart failure, and fibrinolysis were measured in plasma. Pulmonary vessel remodeling was analyzed by immunohistochemistry. RESULTS: Impairing intrathrombus angiogenesis by repeatedly embolizing autologous blood clots containing SU5416 resulted in elevated mean pulmonary arterial pressure (38 mm Hg), increased indexed pulmonary vascular resistance, and enhanced right ventricular hypertrophy (80%, 1.9-fold, 36%, respectively, compared with rabbits embolized with clots containing an antifibrinolytic agent). This was caused by both obstruction of large pulmonary arteries with fibrothrombotic material and muscularization of pulmonary microvessels, and accompanied by inflammatory cell infiltration and increased circulating endothelin-1. CONCLUSIONS: The key role of angiogenesis-driven clot resolution was validated in a reliable small-animal model reproducing the major pathophysiological hallmarks of CTEPH.

Incremental Experience in In Vitro Primary Culture of Human Pulmonary Arterial Endothelial Cells Harvested from Swan-Ganz Pulmonary Arterial Catheters.

Pulmonary arterial hypertension (PAH) is a devastating condition affecting the pulmonary microvascular wall and endothelium, resulting in their partial or total obstruction. Despite a combination of expensive vasodilatory therapies, mortality remains high. Personalized therapeutic approaches, based on access to patient material to unravel patient specificities, could move the field forward. An innovative technique involving harvesting pulmonary arterial endothelial cells (PAECs) at the time of diagnosis was recently described. The aim of the present study was to fine-tune the initial technique and to phenotype the evolution of PAECs in vitro subcultures. PAECs were harvested from Swan-Ganz pulmonary arterial catheters during routine diagnostic or follow up right heart catheterization. Collected PAECs were phenotyped by flow cytometry and immunofluorescence focusing on endothelial-specific markers. We highlight the ability to harvest patients' PAECs and to maintain them for up to 7-12 subcultures. By tracking the endothelial phenotype, we observed that PAECs could maintain an endothelial phenotype for several weeks in culture. The present study highlights the unique opportunity to obtain homogeneous subcultures of primary PAECs from patients at diagnosis and follow-up. In addition, it opens promising perspectives regarding tailored precision medicine for patients suffering from rare pulmonary vascular diseases.

Cytokines trigger disruption of endothelium barrier function and p38 MAP kinase activation in BMPR2-silenced human lung microvascular endothelial cells.

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.

Glyoxalase-1 overexpression partially prevents diabetes-induced impaired arteriogenesis in a rat hindlimb ligation model.

We hypothesize that diabetes-induced impaired collateral formation after a hindlimb ligation in rats is in part caused by intracellular glycation and that overexpression of glyoxalase-I (GLO-I), i.e. the major detoxifying enzyme for advanced-glycation-endproduct (AGE) precursors, can prevent this. Wild-type and GLO-I transgenic rats with or without diabetes (induced by 55 mg/kg streptozotocin) were subjected to ligation of the right femoral artery. Laser Doppler perfusion imaging showed a significantly decreased blood perfusion recovery after 6 days in the diabetic animals compared with control animals, without any effect of Glo1 overexpression. In vivo time-of-flight magnetic resonance angiography at 7-Tesla showed a significant decrease in the number and volume of collaterals in the wild-type diabetic animals compared with the control animals. Glo1 overexpression partially prevented this decrease in the diabetic animals. Diabetes-induced impairment of arteriogenic adaptation can be partially rescued by overexpressing of GLO-I, indicating a role of AGEs in diabetes-induced impaired collateral formation.

Comparison of LDPI to SPECT perfusion imaging using (99m)Tc-sestamibi and (99m)Tc-pyrophosphate in a murine ischemic hind limb model of neovascularization.

BACKGROUND: We aimed to determine the accuracy of laser Doppler perfusion imaging (LDPI) in an animal model for hind limb ischemia. METHODS: We used a murine (C57Bl/6 mice) ischemic hind limb model in which we compared LDPI with the clinically used (99m)Tc-sestamibi SPECT perfusion imaging (n = 7). In addition, we used the SPECT tracer (99m)Tc-pyrophosphate ((99m)Tc-PyP) to image muscular damage (n = 6). RESULTS: LDPI indicated a quick and prominent decrease in perfusion immediately after ligation, subsequently recovering to 21.9 and 25.2 % 14 days later in the (99m)Tc-sestamibi and (99m)Tc-PyP group, respectively. (99m)Tc-sestamibi SPECT scans also showed a quick decrease in perfusion. However, nearly full recovery was reached 7 days post ligation. Muscular damage, indicated by the uptake of (99m)Tc-PyP, was highest at day 3 and recovered to baseline levels at day 14 post ligation. Postmortem histology supported these findings, as a significantly increased collateral diameter was found 7 and 14 days after ligation and peak macrophage infiltration and TUNEL positivity was found on day 3 after ligation. CONCLUSIONS: Here, we indicate that LDPI strongly underestimates perfusion recovery in a hind limb model for profound ischemia.

Nitric Oxide Resistance Reduces Arteriovenous Fistula Maturation in Chronic Kidney Disease in Rats.

BACKGROUND: Autologous 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. METHODS: CKD 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. RESULTS: CKD 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. CONCLUSIONS: CKD 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.

Percutaneous microembolization of the left coronary artery to model ischemic heart disease in rats.

Small animal models of myocardial infarction are used for a wide variety of research purposes, but common techniques for generating such models require thoracic surgeries that increase mortality risk and damage important structures, such as the pericardial sac. Here, we describe a technique for modeling myocardial infarction in rats by selective coronary microembolization, which has hitherto been described only in large animals. This technique selectively catheterizes the left coronary artery using a custom-made catheter that is introduced and precisely placed under fluoroscopic guidance. Microspheres are then injected through the catheter to cause embolization. This process creates multiple simultaneous micro-infarcts that resemble those from clinical embolization after a percutaneous coronary intervention. As this technique does not require thoracic surgery, a low attrition rate was expected and once it was optimized, this technique had a low mortality rate of just 14% during experimental application. This technique creates infarcts that appear small but are associated with transient ECG changes and a persistently lower ejection fraction after embolization. Microspheres are retained in the myocardial tissue and are visible by epifluorescent microscopy after histological staining and recognizable as a distinct speckle pattern in ultrasound images.

CXCL1 microspheres: a novel tool to stimulate arteriogenesis.

CONTEXT: After arterial occlusion, diametrical growth of pre-existing natural bypasses around the obstruction, i.e. arteriogenesis, is the body's main coping mechanism. We have shown before that continuous infusion of chemokine (C-X-C motif) ligand 1 (CXCL1) promotes arteriogenesis in a rodent hind limb ischemia model. OBJECTIVE: For clinical translation of these positive results, we developed a new administration strategy of local and sustained delivery. Here, we investigate the therapeutic potential of CXCL1 in a drug delivery system based on microspheres. MATERIALS AND METHODS: We generated poly(ester amide) (PEA) microspheres loaded with CXCL1 and evaluated them in vitro for cellular toxicity and chemokine release characteristics. In vivo, murine femoral arteries were ligated and CXCL1 was administered either intra-arterially via osmopump or intramuscularly encapsulated in biodegradable microspheres. Perfusion recovery was measured with Laser-Doppler. RESULTS: The developed microspheres were not cytotoxic and displayed a sustained chemokine release up to 28 d in vitro. The amount of released CXCL1 was 100-fold higher than levels in native ligated hind limb. Also, the CXCL1-loaded microspheres significantly enhanced perfusion recovery at day 7 after ligation compared with both saline and non-loaded conditions (55.4 ± 5.0% CXCL1-loaded microspheres versus 43.1 ± 4.5% non-loaded microspheres; n = 8-9; p < 0.05). On day 21 after ligation, the CXCL1-loaded microspheres performed even better than continuous CXCL1 administration (102.1 ± 4.4% CXCL1-loaded microspheres versus 85.7 ± 4.8% CXCL1 osmopump; n = 9; p < 0.05). CONCLUSION: Our results demonstrate a proof of concept that sustained, local delivery of CXCL1 encapsulated in PEA microspheres provides a new tool to stimulate arteriogenesis in vivo.

CXCL1 promotes arteriogenesis through enhanced monocyte recruitment into the peri-collateral space.

AIMS: The mechanisms of monocyte recruitment to arteriogenic collaterals are largely unknown. We investigated the role of chemokine (C-X-C-motif) ligand 1 (CXCL1) and its cognate receptor, chemokine (C-X-C-motif) receptor 2 (CXCR2) in arteriogenesis. METHODS AND RESULTS: After femoral artery ligation in Sprague-Dawley rats, either native collaterals were harvested or placebo, CXCL1 or CXCR2 blocker was administered via an osmopump. Perfusion recovery was measured with Laser Doppler, leukocyte populations were analyzed by fluorescence-activated cell sorting, and hind limb sections were stained for macrophage marker cluster of differentiation 68 (CD68). In vitro, fluorescent CXCL1 or human acute monocytic leukemia cell line (THP-1) monocytic cells were flown over shear-stressed endothelium. CXCL1 mRNA expression in collaterals was dramatically upregulated already 1 h after ligation (ratio ligated/sham 5.73). CD68 mRNA was upregulated from 12 h until 3 days after ligation (peak ratio ligated/sham 2.65). CXCL1 treatment augmented perfusion recovery at 3 and 7 days (p < 0.05) after ligation, and a significant increase in the number of peri-collateral macrophages was evident concomitantly (p < 0.05). Conversely, CXCR2 antagonist treatment caused a decrease in perfusion recovery both at 7 and 10 days postligation (p = 0.01) and also significantly reduced the number of peri-collateral macrophages (p < 0.05). In vitro, CXCL1 tethered to and was taken up by endothelial cells under shear stress conditions and enhanced THP-1 adherence compared to control (p < 0.05). In contrast, CXCR2 antagonist compromised THP-1 adherence to endothelial cells (p < 0.05). CONCLUSION: 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.

Local delivery of polarized macrophages improves reperfusion recovery in a mouse hind limb ischemia model.

AIMS: Enhancement 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 RESULTS: Murine 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 IL10R(fl/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. CONCLUSIONS: Local 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.

Automated multiscale vessel analysis for the quantification of MR angiography of peripheral arteriogenesis.

PURPOSE: To automatically analyze the time course of collateralization in a rat hindlimb ischemia model based on signal intensity distribution (SID). MATERIALS AND METHODS: Time-of-flight magnetic resonance angiograms (TOF-MRA) were acquired in eight rats at 2, 7, and 21 days after unilateral femoral artery ligation. Analysis was performed on maximum intensity projections filtered with multiscale vessel enhancement filter. Differences in SID between ligated limb and a reference region were monitored over time and compared to manual collateral artery identification. RESULTS: The differences in SID correlated well with the number of collateral arteries found with manual quantification. The time courses of ultrasmall (diameter ≪0.5 mm) and small (diameter ≈0.5 mm) collateral artery development could be differentiated, revealing that maturation of the collaterals and enlargement of their feeding arteries occurred mainly after the first week postligation. CONCLUSION: SID analysis performed on axial maximum intensity projections is easy to implement, fast, and objective and provides more insight in the time course of arteriogenesis than manual identification.

Soluble Jagged-1 inhibits neointima formation by attenuating Notch-Herp2 signaling.

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. CONCLUSIONS: 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.

Molecular magnetic resonance imaging of myocardial angiogenesis after acute myocardial infarction.

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.