Acute cardiovascular events, due to rupture or erosion of an atherosclerotic plaque, represent the major cause of morbidity and mortality in patients. Growing evidence suggests that plaque neovascularization is an important contributor to plaque growth and instability. The vessels' immaturity, with profound structural and functional abnormalities, leads to recurrent intraplaque hemorrhage. This review discusses new insights of atherosclerotic neovascularization, including the effects of leaky neovessels on intraplaque hemorrhage, both in experimental models and humans. Furthermore, modalities for in vivo imaging and therapeutic interventions to target plaque angiogenesis will be discussed.

Objective: Statins pleiotropically provide additional benefits in reducing atherosclerosis, but their effects on intraplaque angiogenesis (IPA) and hemorrhage (IPH) remain unclear. Therefore, we discriminated statin’s lipid-lowering dependent and independent effects on IPA and IPH. Approach and results: ApoE3*Leiden mice are statin-responsive due to ApoE and LDLR presence, but also allow to titrate plasma cholesterol levels by diet. Therefore, ApoE3*Leiden mice were fed a high-cholesterol-inducing-diet (HCD) with or without atorvastatin (A) or a moderate-cholesterol-inducing-diet (MCD). Mice underwent vein graft surgery to induce lesions with IPA and IPH. Cholesterol levels were significantly reduced in MCD (56%) and HCD + A (39%) compared to HCD with no significant differences between MCD and HCD + A. Both MCD and HCD + A have a similar reduction in vessel remodeling and inflammation comparing to HCD. IPA was significantly decreased by 30% in HCD + A compared to HCD or MCD. Atorvastatin treatment reduced the presence of immature vessels by 34% vs. HCD and by 25% vs. MCD, resulting in a significant reduction of IPH. Atorvastatin’s anti-angiogenic capacity was further illustrated by a dose-dependent reduction of ECs proliferation and migration. Cultured mouse aortic-segments lost sprouting capacity upon atorvastatin treatment and became 30% richer in VE-Cadherin expression and pericyte coverage. Moreover, Atorvastatin inhibited ANGPT2 release and decreased VE-Cadherin(Y685)-phosphorylation in ECs. Conclusions: Atorvastatin has beneficial effects on vessel remodeling due to its lipid-lowering capacity. Atorvastatin has strong pleiotropic effects on IPA by decreasing the number of neovessels and on IPH by increasing vessel maturation. Atorvastatin improves vessel maturation by inhibiting ANGPT2 release and phospho(Y658)-mediated VE-Cadherin internalization.

Unstable atherosclerotic plaques frequently show plaque angiogenesis which increases the chance of rupture and thrombus formation leading to infarctions. Hypoxia plays a role in angiogenesis and inflammation, two processes involved in the pathogenesis of atherosclerosis. We aim to study the effect of resolution of hypoxia using carbogen gas (95% O₂, 5% CO₂) on the remodeling of vein graft accelerated atherosclerotic lesions in ApoE3*Leiden mice which harbor plaque angiogenesis. Single treatment resulted in a drastic decrease of intraplaque hypoxia, without affecting plaque composition. Daily treatment for three weeks resulted in 34.5% increase in vein graft patency and increased lumen size. However, after three weeks intraplaque hypoxia was comparable to the controls, as were the number of neovessels and the degree of intraplaque hemorrhage. To our surprise we found that three weeks of treatment triggered ROS accumulation and subsequent Hif1a induction, paralleled with a reduction in the macrophage content, pointing to an increase in lesion stability. Similar to what we observed in vivo, in vitro induction of ROS in bone marrow derived macrophages lead to increased Hif1a expression and extensive DNA damage and apoptosis. Our study demonstrates that carbogen treatment did improve vein graft patency and plaque stability and reduced intraplaque macrophage accumulation via ROS mediated DNA damage and apoptosis but failed to have long term effects on hypoxia and intraplaque angiogenesis.

Plaque angiogenesis and plaque hemorrhage are major players in the destabilization and rupture of atherosclerotic lesions. As these are dynamic processes, imaging of plaque angiogenesis, especially the integrity or leakiness of angiogenic vessels, can be an extremely useful tool in the studies on atherosclerosis pathophysiology. Visualizing plaque microvessels in 3D would enable us to study the architecture and permeability of adventitial and intimal plaque microvessels in advanced atherosclerotic lesions. We hypothesized that a comparison of the vascular permeability between healthy continuous and fenestrated as well as diseased leaky microvessels, would allow us to evaluate plaque microvessel leakiness. We developed and validated a two photon intravital microscopy (2P-IVM) method to assess the leakiness of plaque microvessels in murine atherosclerosis-prone ApoE3*Leiden vein grafts based on the quantification of fluorescent-dextrans extravasation in real-time. We describe a novel 2P-IVM set up to study vessels in the neck region of living mice. We show that microvessels in vein graft lesions are in their pathological state more permeable in comparison with healthy continuous and fenestrated microvessels. This 2P-IVM method is a promising approach to assess plaque angiogenesis and leakiness. Moreover, this method is an important advancement to validate therapeutic angiogenic interventions in preclinical atherosclerosis models.