Vasa vasorum lumen narrowing in brain vascular hyalinosis in systemic hypertension patients with ischemic stroke

Ischemic stroke is a major cause of death among patients with systemic hypertension. The narrowing of the lumen of the brain vasculature contributes to the increased incidence of stroke. While hyalinosis represents the major pathological lesions contributing to the vascular lumen narrowing and stroke, the pathogenic mechanism of brain vascular hyalinosis has not been well characterized. Thus, the present study examined the postmortem brain vasculature of human patients who died of ischemic stroke due to systemic hypertension. Hematoxylin and eosin staining and immunohistochemistry showed the occurrence of brain vascular hyalinosis with infiltrated plasma proteins along with the narrowing of vasa vasorum and oxidative stress. Transmission electron microscopy revealed the endothelial cell bulge protrusion into the vasa vasorum lumen and the occurrence of endocytosis in the vasa vasorum endothelium. The treatment of cultured microvascular endothelial cells with adrenaline also promoted the formation of the bulge as well as endocytic vesicles. siRNA knockdown of sortin nexin-9 (a mediator of clathrin-mediated endocytosis) inhibited the adrenaline-induced endothelial cell bulge formation. Adrenaline promoted protein-protein interactions between sortin nexin-9 and neural Wiskott–Aldrich Syndrome protein (a regulator of actin polymerization). We propose that endocytosis-depending endothelial cell bulge narrows the vasa vasorum, resulting in ischemic oxidative damage to the cerebral vessels, the formation of hyalinosis, the occurrence of ischemic stroke, and death in systemic hypertension patients.


Introduction
Stroke is a leading cause of long-term disability and death worldwide. In the United States alone, a stroke occurs every 40 seconds and stroke-induced death occurs every four minutes. Every year, more than 795,000 Americans have a stroke, resulting in 140,000 deaths. [1][2][3] By 2030, an additional 3.4 million U.S. adults are expected to have stroke (20.5% increase in prevalence from 2012). 4,5 About 87% of all stroke incidences are ischemic stroke, which occur when a blood vessel supplying the brain is obstructed. Systemic hypertension is the most important risk factor for the development of ischemic stroke. 1,6 High blood pressure promotes the alterations of the vascular wall and increases the incidence of ischemic stroke and subsequent neurodegeneration. [7][8][9][10] One important lesion that contributes to the lumen narrowing of the brain vessels is vascular hyalinosis, which refers to the thickening of the vascular wall due to deposits of homogeneous hyaline materials. 11,12 The pathogenesis of the formation of hyaline includes the infiltration of plasma proteins such as apolipoprotein E (ApoE), α 2-macroglobulin, fibrinogen, and immunoglobulin G into the vascular wall. 13,14 The accumulation of these plasma components alters all the structural components of the vessel wall through the formation of fibrinoid lesions and causes the hyalinization of the vessel wall. [15][16][17] Thus, the reversal and/or prevention of brain vascular hyalinosis in systemic hypertension patients have the therapeutic potential to reduce the incidence of ischemic stroke. However, the mechanism of brain vascular hyalinosis is not well understood. While the involvement of oxidative stress in various pathological features of stroke has been documented, 18 whether oxidative stress occurs in hyalinosis lesions is unknown. The present study performed detailed histological analyses of the hyalinosis lesions in the postmortem brain tissues of human patients who died of ischemic stroke. We found that the brain vascular hyalinosis in human systemic hypertension patients who died of ischemic stroke is associated with the narrowing of the vasa vasorum that supplies the blood to the cerebral vascular wall and the occurrence of oxidative stress in both the vessel walls as well as the brain tissues.

Histological measurements
The sections were resected to be approximately 10 μ m thick and immersed in 10% buffered formalin at room temperature. The fixed tissue samples were paraffin embedded, sectioned at 6 μ m with a microtome, mounted on glass slides, and analyzed histochemically and immunohistochemically. Tissue sections were subjected to hematoxylin and eosin (H&E) staining to determine the general morphology of the brain vessels, Zerbino-Lukasevich staining to detect the presence of fibrin to assess the vascular lesions, and immunohistochemistry using the anti-ApoE antibody (Abcam, Cambridge, UK) and anti-malondialdehyde (MDA) antibody (Abcam). The vessels smaller than 25 μ m or larger than 150 μ m in external diameter were excluded from analysis.

Transmission Electron Microscopy (TEM)
Brain tissues were collected from patients who underwent neurosurgery and immediately fixed in a solution containing 4% paraformaldehyde and 0.5% glutaraldehyde/0.2 M cacodylate.
Samples were then post-fixed with 1% osmium tetroxide and embedded in EmBed812. Ultrathin sections were stained with uranyl acetate and lead citrate and examined in a Philips EM-400T Transmission Electron Microscope at 80 kV with the TIA software.

Cell culture
Human microvascular endothelial cells were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) and were cultured in accordance with the manufacturer's instructions in 5% CO 2 at 37°C. Cells in passages 3-6 were used.
siRNA experiments were performed using the Santa Cruz Biotechnology (Dallas, TX, USA) system in accordance with the manufacture's instructions. Briefly, cells grown on 6-well plates were incubated with 1 µg siRNA and siRNA Transfection Reagent in siRNA Transfection Medium for 5 hours. Equal volume of growth medium containing 2 times the normal serum, growth supplements and antibiotics were added and cells were grown for 2 days.

Immunoprecipitation and Western blotting
Cell lysates were immunoprecipitated with the mouse polyclonal anti-sortin nexin 9 UltraCruz Autoradiography Films (Santa Cruz Biotechnology). The developed films were scanned and optical densities of protein bands were quantified using NIH ImageJ.

Statistical analysis
Statistical analysis was performed with the IBM SPSS Statistics version 23.0 software.
Significant differences between two groups were determined by the Independent Samples Mann-Whitney U test.

Vasa vasorum narrowing in vascular hyalinosis lesions in the brain of systemic hypertension patients who died of ischemic stroke
Hyalinosis occurs in the brain vasculature in late-stage systemic hypertension preceding ischemic stroke. Hyalinosis is characterized by the death of vascular cells, ultimately leading to the infiltration and accumulation of plasma proteins, forming glass-like materials that occlude the blood vessel lumen and interfere with the gas exchange between the blood and brain tissues.  (Fig. 3).

Endothelial cell bulge protusion into vasa vasorum in the vascular hyalinosis lesions in the brain of systemic hypertension patients who died of ischemic stroke
Our detailed examinations of the hyalinosis lesion in brain tissues collected from human patients who died of ischemic stroke led us to realize that endothelial cell structural changes contribute to the occlusion of the vasa vasorum due to the formation of the "bulge" structure that protrudes from endothelial cells into the lumen of the vasa vasorum ( Fig. 2A). This lead us to formulate a novel hypothesis that the occlusion of this small vessel network supplying the blood to cerebral vascular wall tissues contributes to the death of vascular cells and the formation of hyalinosis. This endothelial cell bulge structure was also visualized by TEM in the vasa vasorum in the hyalinosis lesion of brain vessels of systemic hypertension patients who underwent neurosurgery to remove the hematoma to treat hemorrhagic stroke (Fig. 4A). In the vasa vasorum endothelial cells of brain vasculatures of these patients, we also noted the formation of endocytic vesicles (Fig. 4B).
Also, in cultured human microvascular endothelial cells, the adrenaline treatment promoted the formation of endocytic vesicles as visualized by TEM (Fig. 5A) as well as the bulge-like structure as observed via light microscopy (Fig. 5B). Immunofluorescence staining showed that the bulge formation induced by adrenaline is associated with the reorganization of actin filaments (Fig. 5C). We hypothesized that adrenaline activated the clathrin-mediated endocytosis that, in turn, elicits protein-protein interactions between SNX9 (a mediator of In support of this, we found that siRNA knockdown of SNX9 inhibited the adrenaline-induced bulge formation (Fig. 5B) and that adrenaline promoted SNX9/N-WASp interactions (Fig. 5D).
SHRSP rats exhibited the lesions that are indicative of brain vascular hyalinosis at 8 weeks of age (Fig. 6A). In TEM images of the brain vessels of these rats, we also observed the endothelial cell protrusion into the lumen of vasa vasorum (Fig. 6B) as well as endocytosis in the vasa vasorum endothelial cells (Fig. 6C).

Discussion
In postmortem brain tissues obtained from patients who died of ischemic stroke due to systemic hypertension, we performed histological analyses to define the occurrence of brain vascular hyalinosis. In such lesions, plasma proteins are deposited in the vessel wall and the indication of oxidative stress was detected. We also found that the lumen of the vasa vasorum that supplies the blood to the wall of cerebral vessels was narrowed.
According to our result, lipid peroxidation products reside in both the hyalinosis lesions in the vessels as well as in the brain tissues. Immunohistochemistry using the MDA antibody could stain free MDA or MDA-protein adducts. Thus, it is not yet clear if lipid peroxidation actually occurs in the membrane systems associated with the hyalinosis lesions or the MDAbound protein migrates to the hyalinosis lesions. Plasma proteins that are infiltrated may be oxidized and form oxidation-dependent aggregates, which may then be accumulated as hyaline.
The major finding of this study is that in addition to the cerebral arterial vessels, vasa vasorum is also narrowed. These results may suggest that ischemia-reperfusion injury due to the narrowing of vasa vasorum trigger oxidative stress damage to the cerebral vascular walls, resulting in the subsequent occlusion of the cerebral arteries and ischemic insult to the brain tissue.
The present study provide evidence to support the mechanistic hypothesis as depicted in

Conclusion
In summary, the present study provided histological characterizations of human brain vascular hyalinosis in systemic hypertension patients who died of ischemic stroke. We propose that the vasa vasorum narrowing results in oxidative stress to the cerebral vessels, contributing to the formation of brain vascular hyalinosis, the occurrence of ischemic stroke, further oxidative stress to the neuronal cells, and eventual death in systemic hypertension patients. Further understanding this pathological lesion is critical to developing new and effective therapeutic strategies to reduce the risk of ischemic stroke in systemic hypertension patients, which comprise a large population worldwide.

Funding
This work was supported in part by NIH (R01HL072844, R21AI142649, R03AG059554, and R03AA026516) to Y.J.S. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Conflicts of Interest:
The authors declare no conflict of interest.