mTOR Attenuation with Rapamycin Reverses Neurovascular Uncoupling and Memory Deficits in Mice Modeling Alzheimer's Disease.

Vascular dysfunction is a universal feature of aging and decreased cerebral blood flow has been identified as an early event in the pathogenesis of Alzheimer's disease (AD). Cerebrovascular dysfunction in AD includes deficits in neurovascular coupling (NVC), a mechanism that ensures rapid delivery of energy substrates to active neurons through the blood supply. The mechanisms underlying NVC impairment in AD, however, are not well understood. We have previously shown that mechanistic/mammalian target of rapamycin (mTOR) drives cerebrovascular dysfunction in models of AD by reducing the activity of endothelial nitric oxide synthase (eNOS), and that attenuation of mTOR activity with rapamycin is sufficient to restore eNOS-dependent cerebrovascular function. Here we show mTOR drives NVC impairments in an AD model through the inhibition of neuronal NOS (nNOS)- and non-NOS-dependent components of NVC, and that mTOR attenuation with rapamycin is sufficient to restore NVC and even enhance it above WT responses. Restoration of NVC and concomitant reduction of cortical amyloid-ß levels effectively treated memory deficits in 12-month-old hAPP(J20) mice. These data indicate that mTOR is a critical driver of NVC dysfunction and underlies cognitive impairment in an AD model. Together with our previous findings, the present studies suggest that mTOR promotes cerebrovascular dysfunction in AD, which is associated with early disruption of nNOS activation, through its broad negative impact on nNOS as well as on non-NOS components of NVC. Our studies highlight the potential of mTOR attenuation as an efficacious treatment for AD and potentially other neurologic diseases of aging.SIGNIFICANCE STATEMENT Failure of the blood flow response to neuronal activation [neurovascular coupling (NVC)] in a model of AD precedes the onset of AD-like cognitive symptoms and is driven, to a large extent, by mammalian/mechanistic target of rapamycin (mTOR)-dependent inhibition of nitric oxide synthase activity. Our studies show that mTOR also drives AD-like failure of non-nitric oxide (NO)-mediated components of NVC. Thus, mTOR attenuation may serve to treat AD, where we find that neuronal NO synthase is profoundly reduced early in disease progression, and potentially other neurologic diseases of aging with cerebrovascular dysfunction as part of their etiology.

On P2X receptors in the brain: microvessels. Dedicated to the memory of the late Professor Geoffrey Burnstock (1929-2020).

This tribute article presents selected immunocytochemical and transmission electron microscope data on the location of ATP-gated P2X receptor in the rat brain, as studied in the 1990s in Prof G. Burnstock's laboratory at University College London. There are examples of immuno-ultrastructural findings and introductory information about pre- and post-synaptic location of P2X receptors in the rat cerebellum and endocrine hypothalamus to support the concept of purinergic transmission in the central nervous system. Then findings of diverse immunoreactivity for P2X1, P2X2, P2X4, and P2X6 receptors associated with brain microvessels are shown, including vascular endothelium and pericytes as well as perivascular astrocytes and neuronal components. These findings imply the involvement of P2X receptors and hence purinergic signalling in the neurovascular unit, at least in microvessels in the rat cerebellum and hypothalamic paraventricular and supraoptic nuclei examined here. Various aspects of P2X receptors in brain microvessels are discussed.

Microvasculature in hepatocellular carcinoma: An ultrastructural study.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most vascularized tumor types, and is characterized by development of heterogeneous immature vessels with increased permeability. Here, we analyzed morphology and vascular permeability-related structures in endothelial cells of HCC microvessels. METHODS: Small (Type I) and large (Type II) peritumoral blood microvessels were assessed in HCC-bearing mice. By transmission electron microscopy, endothelial cell cytoplasm area, free transport vesicles, vesiculo-vacuolar organelles and clathrin-coated vesicles were measured. RESULTS: The phenotypic changes in the HCC microvessels included presence of sinusoidal capillarization, numerous luminal microprocesses and abnormal luminal channels, irregular dilatations of interendothelial junctions, local detachment of basement membranes and widened extracellular space. Endothelial cells Type I microvessels showed increased vesicular trafficking-related structures. CONCLUSION: Ultrastructural characteristics of microvessels Type I can associate with HCC new-formed microvessels. The morphological changes observed in HCC microvessels might explain the increased transcellular and paracellular permeability in HCC endothelial cells.

Downregulation of microRNA-103a reduces microvascular endothelial cell injury in a rat model of cerebral ischemia by targeting AXIN2.

Multiple microRNAs (miRNAs) have been found to be linked with cerebral ischemia. Thus, this study was employed to characterize the capabilities of miRNA-103a (miR-103a) on the brain microvascular endothelial cells (BMECs) injury in rat models of middle cerebral artery occlusion (MCAO) by regulating AXIN2. The MCAO rat model was developed by the suture method, where normal saline, miR-103a inhibitors, or its negative control were separately injected into the lateral ventricle to assess the function of miR-103a inhibitors in BMECs apoptosis, microvessel density, as well as angiogenesis. In addition, the oxygen-glucose deprivation model was induced in primarily cultured BMECs to unearth the functions of miR-103a inhibitors on cell viability and apoptosis, lactate dehydrogenase (LDH) release and tube formation ability. Furthermore, the relationship between miR-103a and AXIN2 was verified. The modeled rats of MCAO showed robustly expressed miR-103a, poorly expressed AXIN2, severe neurological deficits, accelerated apoptosis and reduced angiogenesis. miR-103a expression had a negative correlation with AXIN2 messenger RNA expression (r = -0.799; p < .05). In response to the treatment of miR-103a inhibitors, the BMECs apoptosis was suppressed and angiogenesis was restored, corresponding to upregulated Bcl-2, VEGF, and Ang-1, in addition to downregulated caspase-3 and Bax. Meanwhile, AXIN2 was verified to be the miR-103a's target gene. More important, miR-103a inhibitors led to promoted BMEC viability and tube formation and suppressed apoptosis and LDH release rate. This study highlights that miR-103a targets and negatively regulates AXIN2, whereby reducing BMEC injury in cerebral ischemia.

Serial block-face scanning electron microscopy reveals novel intercellular connections in human term placental microvasculature.

The placental microvasculature is a conduit for fetal blood allowing solute exchange between the mother and the fetus. Serial block-face scanning electron microscopy (SBF SEM) allows ultrastructure to be viewed in three dimensions and provides a new perspective on placental anatomy. This study used SBF SEM to study endothelial cells within the human placental microvasculature from uncomplicated pregnancies. Term human placental villi were aldehyde-fixed and processed for imaging by SBF SEM. Manual segmentation was carried out on a terminal villous capillary and an intermediate villous arteriole and venule. Twenty-seven SBF SEM stacks from terminal villi were analysed using stereological approaches to determine the volumes of microvascular components and the proportions of pericyte coverage. SBF SEM analysis of capillary endothelial cells revealed the presence of interendothelial protrusions (IEPs) originating from the donor cell at the endothelial junction and forming deep thin projections up to 7 µm into the adjacent endothelial cells. IEP density was estimated to be in the order of 35 million cm-3 placental tissue. Pericytes cover 15% of the fetal capillary surface area in terminal villi. In comparison, the cytotrophoblast covered 24% of the syncytiotrophoblast basal membrane. A trans-endothelial channel was observed in a region of the vasculo-syncytial capillary. Pericyte coverage was extensive in both arteriole and venule. Three-dimensional imaging of the placental microvasculature identified novel ultrastructural features and provided an insight into factors that may influence capillary permeability and placental function. We hypothesise that the IEPs may allow mechanosensing between adjacent endothelial cells to assist in the maintenance of vessel integrity. The numbers of endothelial junctions, the presence of trans-endothelial channels and the extent of pericyte coverage all provide an insight into the factors determining capillary permeability.

Structural Investigation of Epididymal Microvasculature and Its Relation to Telocytes and Immune Cells in Camel.

The vascular and perivascular cells, including telocytes (TCs) and immune cells, play an important role in male fertility. The current study intended to describe in detail the microvascular structures harboring special regulatory devices in addition to the interstitial cellular components of the one-humped camel epididymis. The samples were collected from 10 clinically healthy mature camels (Camelus dromedarius). The distribution and characteristics of TCs, peripheral blood vessels of the epididymis, and immune cells were investigated using the light, immunohistochemistry, immunofluorescence, and transmission electron microscopy analyses. Frequent occlusive or throttle arterioles were demonstrated in the epididymal interstitium and their tunica media consisted of glomus cells. In addition, some vein walls consisted of one or two layers of glomus cells. TCs, fibroblasts, muscle cells, and tunica media of the blood vessels, that present in the loose connective tissue surrounding the intertubular interstitium of camel epididymis, showed a positive reaction with vimentin. The endothelium of blood vessels and veins showed positive immunoreactivity for CD34 and vascular endothelial growth factor (VEGF). Furthermore, VEGF, CD34, and S100 proteins were expressed in dendritic cells (DCs) as well as TCs. The current data suggest the involvement of DCs and TCs in angiogenesis and a possible role for the interstitial components in creating an appropriate milieu for the full maturation of sperms.

Senescence Alters PPARγ (Peroxisome Proliferator-Activated Receptor Gamma)-Dependent Fatty Acid Handling in Human Adipose Tissue Microvascular Endothelial Cells and Favors Inflammation.

OBJECTIVE: Adipose tissue (AT) dysfunction associated with obesity or aging is a major cause for lipid redistribution and the progression of cardiometabolic disorders. Our goal is to decipher the contribution of human AT microvascular endothelial cells (ECs) in the maintenance of fatty acid (FA) fluxes and the impact of senescence on their function. APPROACH AND RESULTS: We used freshly isolated primary microvascular ECs from human AT. Our data identified the endothelial FA handling machinery including FATPs (FA transport proteins) FATP1, FATP3, FATP4, and CD36 as well as FABP4 (FA binding protein 4). We showed that PPARγ (peroxisome proliferator-activated receptor gamma) regulates the expression of FATP1, CD36, and FABP4 and is a major regulator of FA uptake in human AT EC (hATEC). We provided evidence that endothelial PPARγ activity is modulated by senescence. Indeed, the positive regulation of FA transport by PPARγ agonist was abolished, whereas the emergence of an inflammatory response was favored in senescent hATEC. This was associated with the retention of nuclear FOXO1 (forkhead box protein O1), whereas nuclear PPARγ translocation was impaired. CONCLUSIONS: These data support the notion that PPARγ is a key regulator of primary hATEC function including FA handling and inflammatory response. However, the outcome of PPARγ activation is modulated by senescence, a phenomenon that may impact the ability of hATEC to properly respond to and handle lipid fluxes. Finally, our work highlights the role of hATEC in the regulation of FA fluxes and reveals that dysfunction of these cells with accelerated aging is likely to participate to AT dysfunction and the redistribution of lipids.

Inorganic polyphosphate induces accelerated tube formation of HUVEC endothelial cells.

In this study, the effect of inorganic polyphosphate (polyP) on the initial phase of angiogenesis and vascularization was investigated, applying the HUVEC cell tube formation assay. PolyP is a physiological and high energy phosphate polymer which has been proposed to act as a metabolic fuel in the extracellular space with only a comparably low ATP content. The experiments revealed that polyP accelerates tube formation of human umbilical vein endothelial cells (HUVEC), seeded onto a solidified basement membrane extract matrix which contains polyP-metabolizing alkaline phosphatase (ALP) activity. This effect is abolished by co-addition of apyrase, which degrades ATP to AMP and inorganic phosphate. The assumption that ATP, derived from polyP, activates HUVEC cells leading to tube formation was corroborated by experiments showing that addition of polyP to the cells causes a strong rise of ATP level in the culture medium. Finally, we show that at a later stage of cultivation of HUVEC cells, after 3 d, polyP causes a strong enhancement of the expression of the genes encoding for the two major matrix metalloproteinases (MMPs) released by endothelial cells during tube formation, MMP-9 and MMP-2. This stimulatory effect is again abrogated by addition of apyrase together with polyP. From these results, we propose that polyP is involved either directly or indirectly in energy supply, via ALP-mediated transfer of energy-rich phosphate under ATP formation. This ATP is utilized for the activation and oriented migration of endothelial cells and for the matrix organization during the initial phases of tube formation.

Brain microvascular pathology in Susac syndrome: an electron microscopic study of five cases.

Susac syndrome is a rare, immune-mediated disease characterized by encephalopathy, branch retinal artery occlusion, and hearing loss. Herein, we describe the electron microscopic findings of three brain biopsies and two brain autopsies performed on five patients whose working clinical diagnosis was Susac syndrome. In all five cases, the key findings were basement membrane thickening and collagen deposition in the perivascular space involving small vessels and leading to thickening of vessel walls, narrowing, and vascular occlusion. These findings indicate that Susac syndrome is a microvascular disease. Mononuclear cells were present in the perivascular space, underlining the inflammatory nature of the pathology. Though nonspecific, the changes can be distinguished from genetic and acquired small vessel diseases. The encephalopathy of Susac syndrome overlaps clinically with degenerative and infectious conditions, and brain biopsy may be used for its diagnosis. Its vascular etiology may not be obvious on light microscopy, and electron microscopy is important for its confirmation.

Microvascular Dysfunction Related to Progressive Left Ventricular Remodeling due to Chronic Occlusion of the Left Anterior Descending Artery in an Adult Porcine Heart.

Occlusion of a major coronary artery induces myocardial infarction (MI), leading to left ventricle (LV) remodeling due to progressive microvasculature dysfunction. Irreversible impairment in microvascular function has been suggested to extend from the infarcted region into the infarct-border or remote regions, depending on the time to revascularization. Our aim was to determine whether the occlusion of a major coronary artery induces microvascular dysfunction in the adjacent area perfused by intact coronary arteries using a porcine model for chronic total occlusion of the left anterior descending artery (LAD). MI was induced via an ameroid constrictor ring around the LAD in adult Göttingen pigs (Sus scrofa domesticus, n = 5). Age-matched normal pigs were treated as controls (n = 3). Cardiac magnetic resonance showed reduced systolic regional wall motion in the left circumflex (LCx) and right coronary artery (RCA) territories, with a progressively worsening motion in the infarction-adjacent area over an eight-week period. On 13N-ammonia positron emission tomography (PET), myocardial blood flow (MBF) during hyperemia was significantly greater in the LCx and RCA territories (particularly in the infarction-adjacent area) compared to that in the LAD territory at four weeks after infarct induction. Subsequently, the flow significantly decreased, approaching that in the LAD territory at eight weeks after infarct induction. Fluoroscopy-guided pressure-wire studies showed significantly higher microvascular resistance in the LCx area at eight weeks compared to that in controls. Electron microscopy showed endothelium swelling and microvasculature disruption in areas adjacent to the LCx and RCA territories. Anterior MI caused coronary microvascular dysfunction in the adjacent area, associated with a reduced MBF and regional wall motion.

Direct demonstration of a neonatal Fc receptor (FcRn)-driven endosomal sorting pathway for cellular recycling of albumin.

Albumin is the most abundant plasma protein involved in the transport of many compounds, such as fatty acids, bilirubin, and heme. The endothelial cellular neonatal Fc receptor (FcRn) has been suggested to play a central role in maintaining high albumin plasma levels through a cellular recycling pathway. However, direct mapping of this process is still lacking. This work presents the use of wild-type and engineered recombinant albumins with either decreased or increased FcRn affinity in combination with a low or high FcRn-expressing endothelium cell line to clearly define the FcRn involvement, intracellular pathway, and kinetics of albumin trafficking by flow cytometry, quantitative confocal microscopy, and an albumin-recycling assay. We found that cellular albumin internalization was proportional to FcRn expression and albumin-binding affinity. Albumin accumulation in early endosomes was independent of FcRn-binding affinity, but differences in FcRn-binding affinities significantly affected the albumin distribution between late endosomes and lysosomes. Unlike albumin with low FcRn-binding affinity, albumin with high FcRn-binding affinity was directed less to the lysosomes, suggestive of FcRn-directed albumin salvage from lysosomal degradation. Furthermore, the amount of recycled albumin in cell culture media corresponded to FcRn-binding affinity, with a ∼3.3-fold increase after 1 h for the high FcRn-binding albumin variant compared with wild-type albumin. Together, these findings uncover an FcRn-dependent endosomal cellular-sorting pathway that has great importance in describing fundamental mechanisms of intracellular albumin recycling and the possibility to tune albumin-based therapeutic effects by FcRn-binding affinity.

Novel hemodynamic structures in the human glomerulus.

To investigate human glomerular structure under conditions of physiological perfusion, we have analyzed fresh and perfusion-fixed normal human glomeruli at physiological hydrostatic and oncotic pressures using serial resin section reconstruction, confocal, multiphoton, and electron microscope imaging. Afferent and efferent arterioles (21.5 ± 1.2 µm and 15.9 ± 1.2 µm diameter), recognized from vascular origins, lead into previously undescribed wider regions (43.2 ± 2.8 µm and 38.4 ± 4.9 µm diameter) we have termed vascular chambers (VCs) embedded in the mesangium of the vascular pole. Afferent VC (AVC) volume was 1.6-fold greater than efferent VC (EVC) volume. From the AVC, long nonbranching high-capacity conduit vessels ( n = 7) (Con; 15.9 ± 0.7 µm diameter) led to the glomerular edge, where branching was more frequent. Conduit vessels have fewer podocytes than filtration capillaries. VCs were confirmed in fixed and unfixed specimens with a layer of banded collagen identified in AVC walls by multiphoton and electron microscopy. Thirteen highly branched efferent first-order vessels (E1; 9.9 ± 0.4 µm diameter) converge on the EVC, draining into the efferent arteriole (15.9 ± 1.2 µm diameter). Banded collagen was scarce around EVCs. This previously undescribed branching topology does not conform to the branching of minimum energy expenditure (Murray's law), suggesting that even distribution of pressure/flow to the filtration capillaries is more important than maintaining the minimum work required for blood flow. We propose that AVCs act as plenum manifolds possibly aided by vortical flow in distributing and balancing blood flow/pressure to conduit vessels supplying glomerular lobules. These major adaptations to glomerular capillary structure could regulate hemodynamic pressure and flow in human glomerular capillaries.

Type I interferon causes thrombotic microangiopathy by a dose-dependent toxic effect on the microvasculature.

Many drugs have been reported to cause thrombotic microangiopathy (TMA), yet evidence supporting a direct association is often weak. In particular, TMA has been reported in association with recombinant type I interferon (IFN) therapies, with recent concern regarding the use of IFN in multiple sclerosis patients. However, a causal association has yet to be demonstrated. Here, we adopt a combined clinical and experimental approach to provide evidence of such an association between type I IFN and TMA. We show that the clinical phenotype of cases referred to a national center is uniformly consistent with a direct dose-dependent drug-induced TMA. We then show that dose-dependent microvascular disease is seen in a transgenic mouse model of IFN toxicity. This includes specific microvascular pathological changes seen in patient biopsies and is dependent on transcriptional activation of the IFN response through the type I interferon α/ß receptor (IFNAR). Together our clinical and experimental findings provide evidence of a causal link between type I IFN and TMA. As such, recombinant type I IFN therapies should be stopped at the earliest stage in patients who develop this complication, with implications for risk mitigation.

Ultrastructural changes in microvessels in familial hemiplegic migraine with CACNA1A mutation.

AIMS: Familial hemiplegic migraine type 1 (FHM1) due to mutations in the CACNA1A gene is known as functional vascular disorder with cerebellar atrophy. We describe a case of a FHM1 family in which pathological changes occurred in both brain neuroimaging and skin and muscle biopsy. MATERIALS AND METHODS: In 5 of 18 affected family members, brain MRI scans revealed hyperintense changes in the cerebral white matter. In 2 of these 5 patients, skin and muscle biopsies were performed at the interictal period of the disease and examined under light and transmission electron microscopy. RESULTS: Ultrastructural examination of the biopsy samples revealed abnormal appearance of microvessels resembling oncosis. In the affected vessels, endothelial cells and myocytes/pericytes showed clear cytoplasm, distended endoplasmic reticulum, enlarged mitochondria, and numerous intracytoplasmic vesicular structures. Swollen endothelial cells often significantly narrowed vessel lumen. CONCLUSION: The morphological changes described for the first time in FHM1 suggest that the disease may not only be a functional, but also a structural vascular disorder. We suggest that the presence of these vascular abnormalities can interfere with microcirculation causing damage to the cerebral white matter, visible in MRI scans as hyperintense changes.
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Morphological characterization of pulmonary microvascular disease in bronchopulmonary dysplasia caused by hyperoxia in newborn mice.

PURPOSE: Pulmonary microvascular injury is associated with the pathogenesis of bronchopulmonary dysplasia (BPD). To characterize the mechanisms of pulmonary vascular disease resulting from BPD, we studied the ultrastructural changes affecting pulmonary microvasculature. METHODS: Newborn ICR mice were exposed to 85% hyperoxia or normoxia for 14 days, and then normal air replacement conditions for the following 7 days. At postnatal day (P)14 and P21, lungs were harvested for ultrastructural examination and assessment of pulmonary hypertension. RESULTS: The ultrastructure of pulmonary microvasculature in the hyperoxia-exposed lungs revealed a collapsed capillary lumen. This was due to the abnormal morphology of endothelial cells (ECs) characterized by heterogeneously thick cytoplasm. Compared to normal air controls, the specimens displayed also remarkably thick blood-air barriers (BABs), most of which were occupied by EC layer components. Structural changes were accompanied by increased pulmonary artery medial thickness and right ventricular hypertrophy (RVH). Moreover, abnormalities in ECs persisted even after exposure to 7 days of normal air replacement conditions. Results were confirmed by morphometric quantification. CONCLUSION: Our results suggest that the abnormal morphology of capillary ECs and thick BABs correlates with pulmonary artery remodeling and RVH. These ultrastructural changes might represent possible mechanisms of secondary pulmonary hypertension in BPD.

Sialic acids regulate microvessel permeability, revealed by novel in vivo studies of endothelial glycocalyx structure and function.

KEY POINTS: We have developed novel techniques for paired, direct, real-time in vivo quantification of endothelial glycocalyx structure and associated microvessel permeability. Commonly used imaging and analysis techniques yield measurements of endothelial glycocalyx depth that vary by over an order of magnitude within the same vessel. The anatomical distance between maximal glycocalyx label and maximal endothelial cell plasma membrane label provides the most sensitive and reliable measure of endothelial glycocalyx depth. Sialic acid residues of the endothelial glycocalyx regulate glycocalyx structure and microvessel permeability to both water and albumin. ABSTRACT: The endothelial glycocalyx forms a continuous coat over the luminal surface of all vessels, and regulates multiple vascular functions. The contribution of individual components of the endothelial glycocalyx to one critical vascular function, microvascular permeability, remains unclear. We developed novel, real-time, paired methodologies to study the contribution of sialic acids within the endothelial glycocalyx to the structural and functional permeability properties of the same microvessel in vivo. Single perfused rat mesenteric microvessels were perfused with fluorescent endothelial cell membrane and glycocalyx labels, and imaged with confocal microscopy. A broad range of glycocalyx depth measurements (0.17-3.02 µm) were obtained with different labels, imaging techniques and analysis methods. The distance between peak cell membrane and peak glycocalyx label provided the most reliable measure of endothelial glycocalyx anatomy, correlating with paired, numerically smaller values of endothelial glycocalyx depth (0.078 ± 0.016 µm) from electron micrographs of the same portion of the same vessel. Disruption of sialic acid residues within the endothelial glycocalyx using neuraminidase perfusion decreased endothelial glycocalyx depth and increased apparent solute permeability to albumin in the same vessels in a time-dependent manner, with changes in all three true vessel wall permeability coefficients (hydraulic conductivity, reflection coefficient and diffusive solute permeability). These novel technologies expand the range of techniques that permit direct studies of the structure of the endothelial glycocalyx and dependent microvascular functions in vivo, and demonstrate that sialic acid residues within the endothelial glycocalyx are critical regulators of microvascular permeability to both water and albumin.

Human and mouse cortical astrocytes differ in aquaporin-4 polarization toward microvessels.

Aquaporin-4 (AQP4), the predominant water channel in the brain, is expressed in astrocytes and ependymal cells. In rodents AQP4 is highly polarized to perivascular astrocytic endfeet and loss of AQP4 polarization is associated with disease. The present study was undertaken to compare the expression pattern of AQP4 in human and mouse cortical astrocytes. Cortical tissue specimens were sampled from 11 individuals undergoing neurosurgery wherein brain tissue was removed as part of the procedure, and compared with cortical tissue from 5 adult wild-type mice processed similarly. The tissue samples were immersion-fixed and prepared for AQP4 immunogold electron microscopy, allowing quantitative assessment of AQP4's subcellular distribution. In mouse we found that AQP4 water channels were prominently clustered around vessels, being 5 to 10-fold more abundant in astrocytic endfoot membranes facing the capillary endothelium than in parenchymal astrocytic membranes. In contrast, AQP4 was markedly less polarized in human astrocytes, being only two to three-fold enriched in astrocytic endfoot membranes adjacent to capillaries. The lower degree of AQP4 polarization in human subjects (1/3 of that in mice) was mainly due to higher AQP4 expression in parenchymal astrocytic membranes. We conclude that there are hitherto unrecognized species differences in AQP4 polarization toward microvessels in the cerebral cortex.

Microvascular ultrastructural changes precede cognitive impairment in the murine APPswe/PS1dE9 model of Alzheimer's disease.

Cerebral and systemic organ microvascular pathologies coexist with human Alzheimer's disease (AD) neuropathology. In this study, we hypothesised that both cerebral and systemic microvascular pathologies exist in 4- to 5-month-old male APPswe/PS1dE9 (APP/PS1) transgenic mice prior to the onset of cognitive impairment. To assess this we examined recognition memory in both wild-type and APP/PS1 mice using the object recognition task (ORT; n = 11 per group) and counted thioflavin-S-positive plaques in brain (n = 6 per group). Vascular casts of brain, liver, spleen and kidneys were examined using scanning electron microscopy (n = 6 per group), and the urinary albumin-to-creatinine ratio (uACR; n = 5 per group) was measured as an index of glomerular permeability. Murine recognition memory was intact, as demonstrated by a significant preference for the novel object in the ORT paradigm. Brain sections of wild-type mice were devoid of thioflavin-S positivity, whereas age-matched APP/PS1 mice had an average of 0.88 ± 0.22 thioflavin-S-positive plaques in the cortex, 0.42 ± 0.17 plaques in the dentate gyrus and 0.30 ± 0.07 plaques in the cornus ammonis 1 region. The profiles of casted cerebral capillaries of wild-type mice were smooth and regular in contrast to those of APP/PS1 mice which demonstrate characteristic (0.5-4.6 µm) 'tags'. APP/PS1 mice also had a significantly reduced hepatic vessel number (p = 0.0002) and an increase in the number of splenic microvascular pillars (p = 0.0231), in the absence of changes in either splenic microvascular density (p = 0.3746) or glomerular ultrastructure. The highly significant reduction in uACR in APP/PS1 mice compared to wild-type (p = 0.0079) is consistent with glomerular microvascular dysfunction. These findings highlight early microvascular pathologies in 4- to 5-month-old APP/PS1 transgenic mice and may indicate an amenable target for pharmacological intervention in AD.

Effects of nintedanib on the microvascular architecture in a lung fibrosis model.

Nintedanib, a tyrosine kinase inhibitor approved for the treatment of idiopathic pulmonary fibrosis, has anti-fibrotic, anti-inflammatory, and anti-angiogenic activity. We explored the impact of nintedanib on microvascular architecture in a pulmonary fibrosis model. Lung fibrosis was induced in C57Bl/6 mice by intratracheal bleomycin (0.5 mg/kg). Nintedanib was started after the onset of lung pathology (50 mg/kg twice daily, orally). Micro-computed tomography was performed via volumetric assessment. Static lung compliance and forced vital capacity were determined by invasive measurements. Mice were subjected to bronchoalveolar lavage and histologic analyses, or perfused with a casting resin. Microvascular corrosion casts were imaged by scanning electron microscopy and synchrotron radiation tomographic microscopy, and quantified morphometrically. Bleomycin administration resulted in a significant increase in higher-density areas in the lungs detected by micro-computed tomography, which was significantly attenuated by nintedanib. Nintedanib significantly reduced lung fibrosis and vascular proliferation, normalized the distorted microvascular architecture, and was associated with a trend toward improvement in lung function and inflammation. Nintedanib resulted in a prominent improvement in pulmonary microvascular architecture, which outperformed the effect of nintedanib on lung function and inflammation. These findings uncover a potential new mode of action of nintedanib that may contribute to its efficacy in idiopathic pulmonary fibrosis.

Superior Microvascular Perfusion of Infused Liposome-Encapsulated Hemoglobin Prior to Reductions in Infarctions after Transient Focal Cerebral Ischemia.

BACKGROUND: The development of cerebral infarction after transient ischemia is attributed to postischemic delayed hypoperfusion in the microvascular region. In the present study, we assessed the microvascular perfusion capacity of infused liposome-encapsulated hemoglobin (LEH) in a therapeutic approach for transient middle cerebral artery occlusion (tMCAO). METHODS: Two-hour middle cerebral artery occlusion rats were immediately subjected to intra-arterial infusion of LEH (LEH group) or saline (vehicle group) or no treatment (control group), and then to recanalization. Neurological findings, infarct and edema progression, microvascular endothelial dysfunction, and inflammatory reactions were compared between the 3 groups after 24 hours of reperfusion. Microvascular perfusion in the early phase of reperfusion was evaluated by hemoglobin immunohistochemistry and transmission electron microscopy. RESULTS: The LEH group achieved significantly better results in all items evaluated than the other groups. Hemoglobin immunohistochemistry revealed that the number of hemoglobin-positive microvessels was significantly greater in the LEH group than in the other groups (P < .01), with microvascular perfusion being more likely in narrow microvessels (≤5 µm in diameter). An electron microscopic examination revealed that microvessels in the control group were compressed and narrowed by swollen astrocyte end-feet, whereas those in the LEH group had a less deformed appearance and contained LEH particles and erythrocytes. CONCLUSION: The results of the present study demonstrated that the infusion of LEH reduced infarctions after tMCAO with more hemoglobin-positive and less deformed microvessels at the early phase of reperfusion, suggesting that the superiority of the microvascular perfusion of LEH mediates its neuroprotective effects.