The neurovascular unit plays a central role in numerous cerebral ischemic and degenerative disorders including acute stroke, chronic cerebral hypoxemia, dementia and Alzheimer’s disease. Therefore, processes involved in the maintenance, remodeling and repair of the cerebral vessels are central hubs for numerous disorders and their potential treatment. The Gonzalez Neurovascular Laboratory has been working in concert with clinical and translational research efforts to study the process of angiogenesis in individuals with cerebrovascular steno-occlusive disorders, such as moyamoya disease and intracranial atherosclerosis (ICAS).
Cerebral Steno-Occlusive Disorders and Angiogenesis
Intracranial atherosclerosis is one of the most common causes of stroke worldwide. It accounts for at least 10 percent of all strokes in the United States and as much as 67 percent in countries with predominantly Asian, Hispanic and Black populations. The prognosis for ICAS-caused strokes is worse than for strokes with other etiologies, with an annual rate of recurrent stroke and death of 15 percent despite intensive medical management, and as high as 35 percent in certain populations. Recent randomized controlled clinical trials have shown that angioplasty with stenting and bypass surgery fail to improve outcomes in patients with ICAS.
In the Gonzalez Lab, we have conducted extensive circulating cytokine assessments of patients with ICAS managed medically, and identified circulating profiles associated with failure of medical management. These profiles are characterized by a predominance of antiangiogenic factors endostatin, angiostatin, and VEGFR1, as well as other growth factors previously identified with high risk of stroke, in particular hepatocyte growth factor (HGF). We also have studied, in patients failing medical management and undergoing surgical interventions for revascularization, a cytokine circulating profile associated with reduced neovascularization. This profile, similar to those associated with failure under medical management, is characterized by higher antiangiogenic factors endostatin and angiostatin.
The figures below schematically represent the components of the eigenvector principal component profile associated with failure of medical management and poor neovascularization after synangiosis surgery.
Figure 1. Angiogenic profile (AP4) significantly associated with failure of intensive medical management in patients with ICAS. Notice the predominant components of antiangiogenic factors endostatin, angiostatin, VEGFR1 and the stroke risk predictor HGF. Odds ratio of failure of medical management 7.2 (95%CI= 2.4 – 34.4).
Figure 2. Angiogenic profile significantly (negatively) associated with neovascularization after synangiosis surgery. Notice the predominant components of antiangiogenic factors endostatin and angiostatin (p=0.023).
|Figure 3. Local connected fractal dimension measured using the FracLac plugin for ImageJ in an example of post-synangiosis collaterals. There was a significantly higher mean LCFD in the EDAS collaterals compared with the innate collaterals (1.28 +/- 0.1 versus 1.16 +/- 0.11, P = 0.001). The proportion of high connectivity (LCFD ≥ 1.2) in the entire study population was significantly greater in the EDAS collaterals (P= 0.001) than in the innate collaterals.|
We have also conducted detailed analysis of the neovascularization of patients treated with synangiosis surgery and characterized the patterns of collateral formation in comparison with native collaterals. Interestingly, our evaluations using branching and tortuosity indexes and local connected fractal dimension show that the vessels formed after encephalo-duro-arterio-synangiosis (EDAS) follow branching patterns and tortuosity that resemble mature cerebral vessels. This is contrary to the high tortuosity and low branching patterns of native collaterals.
The clinical application of this work has led to the establishment of EDAS as a valid form of indirect revascularization by synangiosis, in which branches of the external carotid artery (ECA) are rerouted intracranially and placed in intimate contact with middle cerebral artery branches on the brain surface without direct anastomosis. We pioneered this technique and have proven the application of EDAS in adults with moyamoya disease. We are currently conducting a National Institutes of Health-National Institute of Neurological Disorders and Stroke sponsored phase II clinical trial of EDAS in ICAS. Our clinical studies have shown that EDAS produces vascular connections between the ECA branches and the intracranial circulation in ICAS patients and is a promising technique in reducing the risk of stroke despite failure of medical management.
Experimental Model of Synangiosis and Cerebral Ischemia
Having identified candidate angiogenic factors in the treatment of ICAS, we have developed a model of cerebral ischemia in mice by ligation of the distal middle cerebral artery (MCA) (Figure 4).
Figure 4. Mice synangiosis model: A) Animal in custom rotating stereotactic frame – midline incision (dotted line). B) Temporal craniectomy (black arrow) and elevated muscle flap (white arrow). Black dot indicates location for MCA ligation. C) Re-approximated muscle flap in contact with subjacent brain (green arrows).
In the model of synangiosis with MCA ischemia, the Gonzalez Lab has demonstrated increased vascular density in the operative side compared with the control side, as shown in Figure 5. On the left pannel, endothelial cells were marked with CD31. The surgical side exhibits a conglomerate of increased vascular density at the level of the muscle-brain interphase (red line). Colocalization of endothelial cell marker CD31 (red) and nuclear proliferation marker Ki67 (green) showed significant differences between surgical and control sides (73.0 ± 8.3% vs. 33.6 ± 10.9%, p = 0.04), indicating new vessel growth.
Figure 5. Confocal laser microscopy with colocalization of endothelial marker CD31 (red), nuclear marker DAPI (blue) and proliferation marker Ki67 (green), showing proliferation of new vessels.
Targeting Angiogenesis to Foster Protection from Ischemia
Current efforts in the Gonzalez Laboratory are focused on the evaluation of several strategies to stimulate angiogenesis in the setting of cerebral ischemia, in particular through the development of local agents to facilitate vessel formation after synangiosis surgery.