Mice with endothelial cell-selective adhesion molecule deficiency develop coronary microvascular rarefaction and left ventricle diastolic dysfunction.

Endothelial cell-selective adhesion molecule (ESAM) regulates inflammatory cell adhesion and transmigration and promotes angiogenesis. Here, we examined the role of ESAM in cardiac vascularization, inflammatory cell infiltration, and left ventricle (LV) diastolic function under basal and hemodynamic stress conditions. We employed mice with homozygous genetic deletion of ESAM (ESAM-/- ) and also performed uninephrectomy and aldosterone infusion (UNX-Aldo) to induce volume and pressure overload. Using echocardiography, we found that ESAM-/- mice display no change in systolic function. However, they develop LV diastolic dysfunction, as indicated by a significantly reduced E/A ratio (E = early, A = late mitral inflow peak velocities), increased E/e' ratio, isovolumic relaxation time (IVRT), and E wave deceleration time. An unbiased automated tracing and 3D reconstruction of coronary vasculature revealed that ESAM-/- mice had reduced coronary vascular density. Arteries of ESAM-/- mice exhibited impaired endothelial sprouting and in cultured endothelial cells siRNA-mediated ESAM knockdown reduced tube formation. Changes in ESAM-/- mice were accompanied by elevated myocardial inflammatory cytokine and myeloperoxidase-positive neutrophil levels. Furthermore, UNX-Aldo procedure in wild type mice induced LV diastolic dysfunction, which was accompanied by significantly increased serum ESAM levels. When compared to wild types, ESAM-/- mice with UNX-Aldo displayed worsening of LV diastolic function, as indicated by increased IVRT and pulmonary edema. Thus, we propose that ESAM plays a mechanistic role in proper myocardial vascularization and the maintenance of LV diastolic function under basal and hemodynamic stress conditions.

The role of ADAM17 in cerebrovascular and cognitive function in the APP/PS1 mouse model of Alzheimer's disease.

Introduction: The disintegrin and metalloproteinase 17 (ADAM17) exhibits α-secretase activity, whereby it can prevent the production of neurotoxic amyloid precursor protein-α (APP). ADAM17 is abundantly expressed in vascular endothelial cells and may act to regulate vascular homeostatic responses, including vasomotor function, vascular wall morphology, and formation of new blood vessels. The role of vascular ADAM17 in neurodegenerative diseases remains poorly understood. Here, we hypothesized that cerebrovascular ADAM17 plays a role in the pathogenesis of Alzheimer's disease (AD). Methods and results: We found that 9-10 months old APP/PS1 mice with b-amyloid accumulation and short-term memory and cognitive deficits display a markedly reduced expression of ADAM17 in cerebral microvessels. Systemic delivery and adeno-associated virus (AAV)-mediated re-expression of ADAM17 in APP/PS1 mice improved cognitive functioning, without affecting b-amyloid plaque density. In isolated and pressurized cerebral arteries of APP/PS1 mice the endothelium-dependent dilation to acetylcholine was significantly reduced, whereas the vascular smooth muscle-dependent dilation to the nitric oxide donor, sodium nitroprusside was maintained when compared to WT mice. The impaired endothelium-dependent vasodilation of cerebral arteries in APP/PS1 mice was restored to normal level by ADAM17 re-expression. The cerebral artery biomechanical properties (wall stress and elasticity) and microvascular network density was not affected by ADAM17 re-expression in the APP/PS1 mice. Additionally, proteomic analysis identified several differentially expressed molecules involved in AD neurodegeneration and neuronal repair mechanisms that were reversed by ADAM17 re-expression. Discussion: Thus, we propose that a reduced ADAM17 expression in cerebral microvessels impairs vasodilator function, which may contribute to the development of cognitive dysfunction in APP/PS1 mice, and that ADAM17 can potentially be targeted for therapeutic intervention in AD.

Multispectral LEDs Eliminate Lipofuscin-Associated Autofluorescence for Immunohistochemistry and CD44 Variant Detection by in Situ Hybridization in Aging Human, non-Human Primate, and Murine Brain.

A major limitation of mechanistic studies in aging brains is the lack of routine methods to robustly visualize and discriminate the cellular distribution of tissue antigens using fluorescent immunohistochemical multi-labeling techniques. Although such approaches are routine in non-aging brains, they are not consistently feasible in the aging brain due to the progressive accumulation of autofluorescent pigments, particularly lipofuscin, which strongly excite and emit over a broad spectral range. Consequently, aging research has relied upon colorimetric antibody techniques, where discrimination of tissue antigens is often challenging. We report the application of a simple, reproducible, and affordable protocol using multispectral light-emitting diodes (mLEDs) exposure for the reduction/elimination of lipofuscin autofluorescence (LAF) in aging brain tissue from humans, non-human primates, and mice. The mLEDs lamp has a broad spectral range that spans from the UV to infrared range and includes spectra in the violet/blue and orange/red. After photo quenching, the LAF level was markedly reduced when the tissue background fluorescence before and after mLEDs exposure was compared (p < 0.0001) across the spectral range. LAF elimination was estimated at 95 ± 1%. This approach permitted robust specific fluorescent immunohistochemical co-visualization of commonly studied antigens in aging brains. We also successfully applied this method to specifically visualize CD44 variant expression in aging human cerebral white matter using RNAscope fluorescent in-situ hybridization. Photo quenching provides an attractive means to accelerate progress in aging research by increasing the number of molecules that can be topologically discriminated by fluorescence detection in brain tissue from normative or pathological aging.

Association of cerebral microvascular dysfunction and white matter injury in Alzheimer's disease.

Patients with Alzheimer's disease (AD) often have cerebral white matter (WM) hyperintensities on MRI and microinfarcts of presumed microvascular origin pathologically. Here, we determined if vasodilator dysfunction of WM-penetrating arterioles is associated with pathologically defined WM injury and disturbances in quantitative MRI-defined WM integrity in patients with mixed microvascular and AD pathology. We analyzed tissues from 28 serially collected human brains from research donors diagnosed with varying degrees of AD neuropathologic change (ADNC) with or without cerebral microinfarcts (mVBI). WM-penetrating and pial surface arteriolar responses to the endothelium-dependent agonist bradykinin were quantified ex vivo with videomicroscopy. Vascular endothelial nitric oxide synthase (eNOS) and NAD(P)H-oxidase (Nox1, 2 and 4 isoforms) expression were measured with quantitative PCR. Glial fibrillary acidic protein (GFAP)-labeled astrocytes were quantified by unbiased stereological approaches in regions adjacent to the sites of WM-penetrating vessel collection. Post-mortem diffusion tensor imaging (DTI) was used to measure mean apparent diffusion coefficient (ADC) and fractional anisotropy (FA), quantitative indices of WM integrity. In contrast to pial surface arterioles, white matter-penetrating arterioles from donors diagnosed with high ADNC and mVBI exhibited a significantly reduced dilation in response to bradykinin when compared to the other groups. Expression of eNOS was reduced, whereas Nox1 expression was increased in WM arterioles in AD and mVBI cases. WM astrocyte density was increased in AD and mVBI, which correlated with a reduced vasodilation in WM arterioles. Moreover, in cases with low ADNC, bradykinin-induced WM arteriole dilation correlated with lower ADC and higher FA values. Comorbid ADNC and mVBI appear to synergistically interact to selectively impair bradykinin-induced vasodilation in WM-penetrating arterioles, which may be related to reduced nitric oxide- and excess reactive oxygen species-mediated vascular endothelial dysfunction. WM arteriole vasodilator dysfunction is associated with WM injury, as supported by reactive astrogliosis and MRI-defined disrupted WM microstructural integrity.

Role of Caveolae in the Development of Microvascular Dysfunction and Hyperglycemia in Type 2 Diabetes.

In type 2 diabetes (T2D) microvascular dysfunction can interfere with tissue glucose uptake thereby contributing to the development of hyperglycemia. The cell membrane caveolae orchestrate signaling pathways that include microvascular control of tissue perfusion. In this study, we examined the role of caveolae in the regulation of microvascular vasomotor function under the condition of hyperglycemia in T2D patients and rodent models. Human coronary arterioles were obtained during cardiac surgery from T2D patients, with higher perioperative glucose levels, and from normoglycemic, non-diabetic controls. The coronary arteriole responses to pharmacological agonists bradykinin and acetylcholine were similar in T2D and non-diabetic patients, however, exposure of the isolated arteries to methyl-ß-cyclodextrin (mßCD), an agent known to disrupt caveolae, reduced vasodilation to bradykinin selectively in T2D subjects and converted acetylcholine-induced vasoconstriction to dilation similarly in the two groups. Dilation to the vascular smooth muscle acting nitric oxide donor, sodium nitroprusside, was not affected by mßCD in either group. Moreover, mßCD reduced endothelium-dependent arteriolar dilation to a greater extent in hyperglycemic and obese db/db mice than in the non-diabetic controls. Mechanistically, when fed a high-fat diet (HFD), caveolin-1 knockout mice, lacking caveolae, exhibited a significantly reduced endothelium-dependent arteriolar dilation, both ex vivo and in vivo, which was accompanied by significantly higher serum glucose levels, when compared to HFD fed wild type controls. Thus, in T2D arterioles the role of caveolae in regulating endothelium-dependent arteriole dilation is altered, which appears to maintain vasodilation and mitigate the extent of hyperglycemia. While caveolae play a unique role in microvascular vasomotor regulation, under the condition of hyperglycemia arterioles from T2D subjects appear to be more susceptible for caveolae disruption-associated vasomotor dysfunction and impaired glycemic control.

Aging-induced impaired endothelial wall shear stress mechanosensing causes arterial remodeling via JAM-A/F11R shedding by ADAM17.

Physiological and pathological vascular remodeling is uniquely driven by mechanical forces from blood flow in which wall shear stress (WSS) mechanosensing by the vascular endothelium plays a pivotal role. This study aimed to determine the novel role for a disintegrin and metalloproteinase 17 (ADAM17) in impaired WSS mechanosensing, which was hypothesized to contribute to aging-associated abnormal vascular remodeling. Without changes in arterial blood pressure and blood flow rate, skeletal muscle resistance arteries of aged mice (30-month-old vs. 12-week-old) exhibited impaired WSS mechanosensing and displayed inward hypertrophic arterial remodeling. These vascular changes were recapitulated by in vivo confined, AAV9-mediated overexpression of ADAM17 in the resistance arteries of young mice. An aging-related increase in ADAM17 expression reduced the endothelial junction level of its cleavage substrate, junctional adhesion molecule-A/F11 receptor (JAM-A/F11R). In cultured endothelial cells subjected to steady WSS ADAM17 activation or JAM-A/F11R knockdown inhibited WSS mechanosensing. The ADAM17-activation induced, impaired WSS mechanosensing was normalized by overexpression of ADAM17 cleavage resistant, mutated JAM-AV232Y both in cultured endothelial cells and in resistance arteries of aged mice, in vivo. These data demonstrate a novel role for ADAM17 in JAM-A/F11R cleavage-mediated impaired endothelial WSS mechanosensing and subsequently developed abnormal arterial remodeling in aging. ADAM17 could prove to be a key regulator of WSS mechanosensing, whereby it can also play a role in pathological vascular remodeling in diseases.

Coronary Microvascular Dysfunction and Heart Failure with Preserved Ejection Fraction - implications for Chronic Inflammatory Mechanisms.

Coronary Microvascular Dysfunction (CMD) is now considered one of the key underlying pathologies responsible for the development of both acute and chronic cardiac complications. It has been long recognized that CMD contributes to coronary no-reflow, which occurs as an acute complication during percutaneous coronary interventions. More recently, CMD was proposed to play a mechanistic role in the development of left ventricle diastolic dysfunction in heart failure with preserved ejection fraction (HFpEF). Emerging evidence indicates that a chronic low-grade pro-inflammatory activation predisposes patients to both acute and chronic cardiovascular complications raising the possibility that pro-inflammatory mediators serve as a mechanistic link in HFpEF. Few recent studies have evaluated the role of the hyaluronan-CD44 axis in inflammation-related cardiovascular pathologies, thus warranting further investigations. This review article summarizes current evidence for the role of CMD in the development of HFpEF, focusing on molecular mediators of chronic proinflammatory as well as oxidative stress mechanisms and possible therapeutic approaches to consider for treatment and prevention.