Nanoliposomes Reduce Stroke Injury Following Middle Cerebral Artery Occlusion in Mice.

BACKGROUND AND PURPOSE: Neuroprotective strategies for stroke remain inadequate. Nanoliposomes comprised of phosphatidylcholine, cholesterol, and monosialogangliosides (nanoliposomes) induced an antioxidant protective response in endothelial cells exposed to amyloid insults. We tested the hypotheses that nanoliposomes will preserve human neuroblastoma (SH-SY5Y) and human brain microvascular endothelial cells viability following oxygen-glucose deprivation (OGD)-reoxygenation and will reduce injury in mice following middle cerebral artery occlusion. METHODS: SH-SY5Y and human brain microvascular endothelial cells were exposed to oxygen-glucose deprivation-reoxygenation (3 hours 0.5%-1% oxygen and glucose-free media followed by 20-hour ambient air/regular media) without or with nanoliposomes (300 µg/mL). Viability was measured (calcein-acetoxymethyl fluorescence) and protein expression of antioxidant proteins HO-1 (heme oxygenase-1), NQO1 (NAD[P]H quinone dehydrogenase 1), and SOD1 (superoxide dismutase 1) were measured by Western blot. C57BL/6J mice were treated with saline (n=8) or nanoliposomes (10 mg/mL lipid, 200 µL, n=7) while undergoing 60-minute middle cerebral artery occlusion followed by reperfusion. Day 2 postinjury neurological impairment score and infarction size were compared. RESULTS: SH-SY5Y and human brain microvascular endothelial cells showed reduced viability post-oxygen-glucose deprivation-reoxygenation that was reversed by nanoliposomes. Nanoliposomes increased protein expressions of HO-1, NQO1 in both cell types and SOD1 in human brain microvascular endothelial cells. Nanoliposomes-treated mice showed reduced neurological impairment and brain infarct size (18.8±2% versus 27.3±2.3%, P=0.017) versus controls. CONCLUSIONS: Nanoliposomes reduced stroke injury in mice subjected to middle cerebral artery occlusion likely through induction of an antioxidant protective response. Nanoliposome is a candidate novel agent for stroke.

Human cerebral collateral arteriole function in subjects with normal cognition, mild cognitive impairment, and dementia.

Clinical and preclinical studies have suggested a link between cardiovascular disease and dementia disorders, but the role of the collateral brain circulation in cognitive dysfunction remains unknown. We aimed to test the hypothesis that leptomeningeal arteriole (LMA) function and response to metabolic stressors differ among subjects with dementia, mild cognitive impairment (MCI), and normal cognition (CN). After rapid autopsy, LMAs were isolated from subjects with CN ( n = 10), MCI ( n = 12), or dementia [ n = 42, Alzheimer's disease (AD), vascular dementia (VaD), or other dementia], and endothelial and smooth muscle-dependent function were measured at baseline and after exposure to ß-amyloid (2 µM), palmitic acid (150 µM), or medin (5 µM) and compared. There were no differences among the groups in baseline endothelial function (maximum dilation to acetylcholine, CN: 74.1 ± 9.7%, MCI: 67.1 ± 4.8%, AD: 74.7 ± 2.8%, VaD: 72.0 ± 5.3%, and other dementia: 68.0 ± 8.0%) and smooth muscle-dependent function (CN: 93.4 ± 3.0%, MCI: 83.3 ± 4.1%, AD: 91.8 ± 1.7%, VaD: 91.7 ± 2.4%, and other dementia: 87.9 ± 4.9%). There was no correlation between last cognitive function score and baseline endothelial or smooth muscle-dependent function. LMA endothelial function and, to a lesser extent, smooth muscle-dependent function were impaired posttreatment with ß-amyloid, palmitic acid, and medin. Posttreatment LMA responses were not different between subjects with CN/MCI vs. dementia. Baseline responses and impaired vasoreactivity after treatment with metabolic stressors did not differ among subjects with CN, MCI, and dementia. The results suggest that the cognitive dysfunction in dementia disorders is not attributable to differences in baseline brain collateral circulation function but may be influenced by exposure of the vasculature to metabolic stressors.

PEGylated-nanoliposomal clusterin for amyloidogenic light chain-induced endothelial dysfunction.

Light chain (AL) amyloidosis is a disease associated with significant morbidity and mortality arising from multi-organ injury induced by amyloidogenic light chain proteins (LC). There is no available treatment to reverse the toxicity of LC. We previously showed that chaperone glycoprotein clusterin (CLU) and nanoliposomes (NL), separately, restore human microvascular endothelial function impaired by LC. In this work, we aim to prepare PEGylated-nanoliposomal clusterin (NL-CLU) formulations that could allow combined benefit against LC while potentially enabling efficient delivery to microvascular tissue, and test efficacy on human arteriole endothelial function. NL-CLU was prepared by a conjugation reaction between the carboxylated surface of NL and the primary amines of the CLU protein. NL were made of phosphatidylcholine (PC), cholesterol (Chol) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG 2000 carboxylic acid) at 70:25:5 mol%. The protective effect of NL-CLU was tested by measuring the dilation response to acetylcholine and papaverine in human adipose arterioles exposed to LC. LC treatment significantly reduced the dilation response to acetylcholine and papaverine; co-treatment of LC with PEGylated-nanoliposomal CLU or free CLU restored the dilator response. NL-CLU is a feasible and promising approach to reverse LC-induced endothelial damage.

Medin Induces Pro-Inflammatory Activation of Human Brain Vascular Smooth Muscle Cells.

Background: Medin is one of the most common amyloidogenic proteins and accumulates in the vasculature with aging. Vascular medin accumulation is associated with Alzheimer's disease, vascular dementia and aortic aneurysms. Medin impairs smooth muscle-dependent vasodilation in isolated human brain cerebral arteries. The role of medin in vascular smooth muscle (VSMC) activation is unknown. We aim to evaluate the effects of medin on human brain VSMC activation. Methods: VSMCs were exposed to physiologic doses of medin (0.5, 1 and 5 µM) without or with small molecule nuclear factor-κB (NFκB) inhibitor RO106-9920 (10 µM) for 20 hours. Polymerase chain reaction, Western blot/enzyme-linked immunosorbent assay were used to quantify gene and protein expressions/secretions, respectively, of pro-inflammatory factors (interleukin (IL)-6, IL-8 and monocyte chemoattractant protein (MCP)-1) and structural and enzyme proteins associated with VSMC phenotypic transformation (smooth muscle actin alpha 2 (ACTA2), myosin heavy chain 11 (MYH11) and NADPH oxidase 4 (NOX4)). Results: Medin exposure increased VSMC gene expression and protein secretion of IL-6, IL-8 and MCP-1 (protein secretion 46.0±12.8x, 20.2±4.1x and 8.7±3.1x, respectively, medin 5 µM versus vehicle, all p<0.05). There was no change in gene or protein expressions of ACTA2, MYH11 and NOX4. Co-treatment with RO106-9920 reduced medin-induced increases in IL-6 and IL-8 and a trend towards reduced MCP-1 secretion. Conclusions: Medin induced pro-inflammatory activation of human brain VSMCs that is mediated, at least in part, by NFκB. Acute medin treatment did not alter structural proteins involved in VSMC phenotypic transformation. The findings support medin as a potential novel mediator of and therapeutic target for vascular aging pathology.

Engineered Gold and Silica Nanoparticle-Incorporated Hydrogel Scaffolds for Human Stem Cell-Derived Cardiac Tissue Engineering.

Electrically conductive biomaterials and nanomaterials have demonstrated great potential in the development of functional and mature cardiac tissues. In particular, gold nanomaterials have emerged as promising candidates due to their biocompatibility and ease of fabrication for cardiac tissue engineering utilizing rat- or stem cell-derived cardiomyocytes (CMs). However, despite significant advancements, it is still not clear whether the enhancement in cardiac tissue function is primarily due to the electroconductivity features of gold nanoparticles or the structural changes of the scaffold resulting from the addition of these nanoparticles. To address this question, we developed nanoengineered hydrogel scaffolds comprising gelatin methacrylate (GelMA) embedded with either electrically conductive gold nanorods (GNRs) or nonconductive silica nanoparticles (SNPs). This enabled us to simultaneously assess the roles of electrically conductive and nonconductive nanomaterials in the functionality and fate of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Our studies revealed that both GNR- and SNP-incorporated hydrogel scaffolds exhibited excellent biocompatibility and similar cardiac cell attachment. Although the expression of sarcomere alpha-actinin did not significantly differ among the conditions, a more organized sarcomere structure was observed within the GNR-embedded hydrogels compared to the nonconductive nanoengineered scaffolds. Furthermore, electrical coupling was notably improved in GNR-embedded scaffolds, as evidenced by the synchronous calcium flux and enhanced calcium transient intensity. While we did not observe a significant difference in the gene expression profile of human cardiac tissues formed on the conductive GNR- and nonconductive SNP-incorporated hydrogels, we noticed marginal improvements in the expression of some calcium and structural genes in the nanomaterial-embedded hydrogel groups as compared to the control condition. Given that the cardiac tissues formed atop the nonconductive SNP-based scaffolds (used as the control for conductivity) also displayed similar levels of gene expression as compared to the conductive hydrogels, it suggests that the electrical conductivity of nanomaterials (i.e., GNRs) may not be the sole factor influencing the function and fate of hiPSC-derived cardiac tissues when cells are cultured atop the scaffolds. Overall, our findings provide additional insights into the role of electrically conductive gold nanoparticles in regulating the functionalities of hiPSC-CMs.

Transcriptomic analyses reveal proinflammatory activation of human brain microvascular endothelial cells by aging-associated peptide medin and reversal by nanoliposomes.

Medin is a common vascular amyloidogenic peptide recently implicated in Alzheimer's disease (AD) and vascular dementia and its pathology remains unknown. We aim to identify changes in transcriptomic profiles and pathways in human brain microvascular endothelial cells (HBMVECs) exposed to medin, compare that to exposure to ß-amyloid (Aß) and evaluate protection by monosialoganglioside-containing nanoliposomes (NL). HBMVECs were exposed for 20 h to medin (5 µM) without or with Aß(1-42) (2 µM) or NL (300 µg/mL), and RNA-seq with signaling pathway analyses were performed. Separately, reverse transcription polymerase chain reaction of select identified genes was done in HBMVECs treated with medin (5 µM) without or with NFκB inhibitor RO106-9920 (10 µM) or NL (300 µg/mL). Medin caused upregulation of pro-inflammatory genes that was not aggravated by Aß42 co-treatment but reversed by NL. Pathway analysis on differentially expressed genes revealed multiple pro-inflammatory signaling pathways, such as the tumor necrosis factor (TNF) and the nuclear factor-κB (NFkB) signaling pathways, were affected specifically by medin treatment. RO106-9920 and NL reduced medin-induced pro-inflammatory activation. Medin induced endothelial cell pro-inflammatory signaling in part via NFκB that was reversed by NL. This could have potential implications in the pathogenesis and treatment of vascular aging, AD and vascular dementia.

Modeling long QT syndrome type 2 on-a-chip via in-depth assessment of isogenic gene-edited 3D cardiac tissues.

Long QT syndrome (LQTS) is a cardiovascular disease characterized by QT interval prolongation that can lead to sudden cardiac death. Many mutations with heterogeneous mechanisms have been identified in KCNH2, the gene that encodes for hERG (Kv11.1), which lead to onset of LQTS type 2 (LQTS2). In this work, we developed a LQTS2-diseased tissue-on-a-chip model, using 3D coculture of isogenic stem cell-derived cardiomyocytes (CMs) and cardiac fibroblasts (CFs) within an organotypic microfluidic chip technology. Primarily, we created a hiPSC line with R531W mutation in KCNH2 using CRISPR-Cas9 gene-editing technique and characterized the resultant differentiated CMs and CFs. A deficiency in hERG trafficking was identified in KCNH2-edited hiPSC-CMs, revealing a possible mechanism of R531W mutation in LQTS2 pathophysiology. Following creation of a 3D LQTS2 tissue-on-a-chip, the tissues were extensively characterized, through analysis of calcium handling and response to ß-agonist. Furthermore, attempted phenotypic rescue via pharmacological intervention of LQTS2 on a chip was investigated.

Chronic Cognitive and Cerebrovascular Function after Mild Traumatic Brain Injury in Rats.

Severe traumatic brain injury (TBI) results in cognitive dysfunction in part due to vascular perturbations. In contrast, the long-term vasculo-cognitive pathophysiology of mild TBI (mTBI) remains unknown. We evaluated mTBI effects on chronic cognitive and cerebrovascular function and assessed their interrelationships. Sprague-Dawley rats received midline fluid percussion injury (n = 20) or sham (n = 21). Cognitive function was assessed (3- and 6-month novel object recognition [NOR], novel object location [NOL], and temporal order object recognition [TOR]). Six-month cerebral blood flow (CBF) and cerebral blood volume (CBV) using contrast magnetic resonance imaging (MRI) and ex vivo circle of Willis artery endothelial and smooth muscle-dependent function were measured. mTBI rats showed significantly impaired NOR, with similar trends (non-significant) in NOL/TOR. Regional CBF and CBV were similar in sham and mTBI. NOR correlated with CBF in lateral hippocampus, medial hippocampus, and primary somatosensory barrel cortex, whereas it inversely correlated with arterial smooth muscle-dependent dilation. Six-month baseline endothelial and smooth muscle-dependent arterial function were similar among mTBI and sham, but post-angiotensin 2 stimulation, mTBI showed no change in smooth muscle-dependent dilation from baseline response, unlike the reduction in sham. mTBI led to chronic cognitive dysfunction and altered angiotensin 2-stimulated smooth muscle-dependent vasoreactivity. The findings of persistent pathophysiological consequences of mTBI in this animal model add to the broader understanding of chronic pathophysiological sequelae in human mild TBI.

Cerebrovascular medin is associated with Alzheimer's disease and vascular dementia.

INTRODUCTION: Medin, an aging-associated amyloidogenic protein, induces cerebrovascular dysfunction and inflammation. We investigated the relationship between cerebrovascular medin and Alzheimer's disease (AD) and vascular dementia (VaD). METHODS: Cerebral arteriole medin was quantified from 91 brain donors with no dementia (ND), AD, VaD, or combined AD and VaD. Correlation analyses evaluated the relationship between arteriole medin, and plaques, tangles, or white matter lesions (WML). Receiver operating characteristic and regression analyses assessed whether medin is predictive of AD or VaD versus other cerebrovascular pathologies (circle of Willis [CoW] atherosclerosis and cerebral amyloid angiopathy [CAA]). RESULTS: Arteriole medin was higher in those with AD, VaD, or combined AD/VaD versus ND (P < .05), and correlated with tangle, plaque, and WML, but not CAA or CoW atherosclerosis. Among cerebrovascular pathologies, medin was the strongest predictor of AD diagnosis, whereas CoW atherosclerosis and arteriole medin were predictors of VaD. DISCUSSION: Cerebral arteriole medin is associated with and could be a potential novel risk factor or biomarker for AD and VaD.

Endothelial Immune Activation by Medin: Potential Role in Cerebrovascular Disease and Reversal by Monosialoganglioside-Containing Nanoliposomes.

Background The function of medin, one of the most common human amyloid proteins that accumulates in the vasculature with aging, remains unknown. We aim to probe medin's role in cerebrovascular disease by comparing cerebral arterial medin content between cognitively normal and vascular dementia (VaD) patients and studying its effects on endothelial cell (EC) immune activation and neuroinflammation. We also tested whether monosialoganglioside-containing nanoliposomes could reverse medin's adverse effects. Methods and Results Cerebral artery medin and astrocyte activation were measured and compared between VaD and cognitively normal elderly brain donors. ECs were exposed to physiologic dose of medin (5 µmol/L), and viability and immune activation (interleukin-8, interleukin-6, intercellular adhesion molecule-1, and plasminogen activator inhibitor-1) were measured without or with monosialoganglioside-containing nanoliposomes (300 µg/mL). Astrocytes were exposed to vehicle, medin, medin-treated ECs, or their conditioned media, and interleukin-8 production was compared. Cerebral collateral arterial and parenchymal arteriole medin, white matter lesion scores, and astrocyte activation were higher in VaD versus cognitively normal donors. Medin induced EC immune activation (increased interleukin-8, interleukin-6, intercellular adhesion molecule-1, and plasminogen activator inhibitor-1) and reduced EC viability, which were reversed by monosialoganglioside-containing nanoliposomes. Interleukin-8 production was augmented when astrocytes were exposed to medin-treated ECs or their conditioned media. Conclusions Cerebral arterial medin is higher in VaD compared with cognitively normal patients. Medin induces EC immune activation that modulates astrocyte activation, and its effects are reversed by monosialoganglioside-containing nanoliposomes. Medin is a candidate novel risk factor for aging-related cerebrovascular disease and VaD.

Amyloidogenic medin induces endothelial dysfunction and vascular inflammation through the receptor for advanced glycation endproducts.

AIMS: Medin is a common amyloidogenic protein in humans that accumulates in arteries with advanced age and has been implicated in vascular degeneration. Medin's effect on endothelial function remains unknown. The aims are to assess medin's effects on human arteriole endothelial function and identify potential mechanisms underlying medin-induced vascular injury. METHODS AND RESULTS: Ex vivo human adipose and leptomeningeal arterioles were exposed (1 h) to medin (0.1, 1, or 5 µM) without or with FPS-ZM1 [100 µM, receptor for advanced glycation endproducts (RAGE)-specific inhibitor] and endothelium-dependent function (acetylcholine dilator response) and endothelium-independent function (dilator response to nitric oxide donor diethylenetriamine NONOate) were compared with baseline control. Human umbilical vein endothelial cells were exposed to medin without or with FPS-ZM1 and oxidative and nitrative stress, cell viability, and pro-inflammatory signaling measures were obtained. Medin caused impaired endothelial function (vs. baseline response: -45.2 ± 5.1 and -35.8 ± 7.9% in adipose and leptomeningeal arterioles, respectively, each P < 0.05). Dilator response to NONOate was not significantly changed. Medin decreased arteriole and endothelial cell nitric oxide production, increased superoxide production, reduced endothelial cell viability, proliferation, and migration. Medin increased gene and protein expression of interleukin-6 and interleukin-8 via activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB). Medin-induced endothelial dysfunction and oxidative stress were reversed by antioxidant polyethylene glycol superoxide dismutase and by RAGE inhibitor FPS-ZM1. CONCLUSIONS: Medin causes human microvascular endothelial dysfunction through oxidative and nitrative stress and promotes pro-inflammatory signaling in endothelial cells. These effects appear to be mediated via RAGE. The findings represent a potential novel mechanism of vascular injury.

Nanoliposomes protect against human arteriole endothelial dysfunction induced by ß-amyloid peptide.

We tested whether nanoliposomes containing phosphatidylcholine, cholesterol and phosphatidic acid (NLPA) prevent ß-amyloid 1-42 (Aß42) fibrillation and Aß42-induced human arteriole endothelial dysfunction. NLPA abolished Aß42 fibril formation (thioflavin-T fluorescence/electron microscopy). In ex-vivo human adipose and leptomeningeal arterioles, Aß42 impaired dilator response to acetylcholine that was reversed by NLPA; this protection was abolished by L-NG-nitroarginine methyl ester. Aß42 reduced human umbilical vein endothelial cell NO production that was restored by NLPA. Nanoliposomes prevented Aß42 amyloid formation, reversed Aß42-induced human microvascular endothelial dysfunction and may be useful in Alzheimer's disease.

Monosialoganglioside-Containing Nanoliposomes Restore Endothelial Function Impaired by AL Amyloidosis Light Chain Proteins.

BACKGROUND: Light chain amyloidosis (AL) is associated with high mortality, especially in patients with advanced cardiovascular involvement. It is caused by toxicity of misfolded light chain proteins (LC) in vascular, cardiac, and other tissues. There is no treatment to reverse LC tissue toxicity. We tested the hypothesis that nanoliposomes composed of monosialoganglioside, phosphatidylcholine, and cholesterol (GM1 ganglioside-containing nanoliposomes [NLGM1]) can protect against LC-induced human microvascular dysfunction and assess mechanisms behind the protective effect. METHODS AND RESULTS: The dilator responses of ex vivo abdominal adipose arterioles from human participants without AL to acetylcholine and papaverine were measured before and after exposure to LC (20 µg/mL) with or without NLGM1 (1:10 ratio for LC:NLGM1 mass). Human umbilical vein endothelial cells were exposed for 18 to 20 hours to vehicle, LC with or without NLGM1, or NLGM1 and compared for oxidative and nitrative stress response and cellular viability. LC impaired arteriole dilator response to acetylcholine, which was restored by co-treatment with NLGM1. LC decreased endothelial cell nitric oxide production and cell viability while increasing superoxide and peroxynitrite; these adverse effects were reversed by NLGM1. NLGM1 increased endothelial cell protein expression of antioxidant enzymes heme oxygenase 1 and NAD(P)H quinone dehydrogenase 1 and increased nuclear factor, erythroid 2 like 2 (Nrf-2) protein. Nrf-2 gene knockdown reduced antioxidant stress response and reversed the protective effects of NLGM1. CONCLUSIONS: NLGM1 protects against LC-induced human microvascular endothelial dysfunction through increased nitric oxide bioavailability and reduced oxidative and nitrative stress mediated by Nrf-2-dependent antioxidant stress response. These findings point to a potential novel therapeutic approach for light chain amyloidosis.