Capillary pathology with prominent basement membrane reduplication is the hallmark histopathological feature of scleromyositis.

AIMS: We aim to perform ultrastructural and histopathological analysis of muscle biopsies from a large group of systemic sclerosis (SSc) patients, including some with early/mild SSc features, and examine whether capillary pathology differentiates 'scleromyositis' (SM) from other auto-immune myositis (AIM) subsets. METHODS: Muscle biopsies from a total of 60 SM patients and 43 AIM controls from two independent cohorts were examined by electron microscopy, collagen-4 immunofluorescence (Col4IF) and routine light microscopy. RESULTS: Ultrastructural examination revealed prominent capillary basement membrane (BM) reduplication (4+ layers in >50% of capillaries) in 65% of SM vs 0% of AIM controls (p < 0.001). In SM cases without prominent BM reduplication, capillary dilation was the most distinctive feature, present in 8% of capillaries in SM vs 2% in controls (p = 0.001). Accumulation of ensheathed pericyte processes was another characteristic feature of SM and closely correlated with the degree of BM reduplication (r = 0.833, p < 0.001). On light microscopy, BM marker Col4IF revealed more frequent capillary enlargement in SM than in controls (84% vs 21%, p < 0.001). SM cases were classified as non-inflammatory myopathy (36%), non-specific myositis (33%) or immune-mediated necrotizing myopathy (31%), but despite this histopathological heterogeneity, prominent BM reduplication remained a constant finding. In the 16 SM patients with early/mild SSc features, 63% showed prominent BM reduplication. CONCLUSIONS: These results show that capillary pathology, and in particular prominent capillary BM reduplication, is the hallmark histopathological feature of SM even in patients with early/mild SSc and support the concept of SM as an organ manifestation of SSc and a distinct subset of AIM.

A Developmental Analysis of Juxtavascular Microglia Dynamics and Interactions with the Vasculature.

Microglia, a resident CNS macrophage, are dynamic cells, constantly extending and retracting their processes as they contact and functionally regulate neurons and other glial cells. There is far less known about microglia-vascular interactions, particularly under healthy steady-state conditions. Here, we use the male and female mouse cerebral cortex to show that a higher percentage of microglia associate with the vasculature during the first week of postnatal development compared with older ages and that the timing of these associations is dependent on the fractalkine receptor (CX3CR1). Similar developmental microglia-vascular associations were detected in the human brain. Using live imaging in mice, we found that juxtavascular microglia migrated when microglia are actively colonizing the cortex and became stationary by adulthood to occupy the same vascular space for nearly 2 months. Further, juxtavascular microglia at all ages associate with vascular areas void of astrocyte endfeet, and the developmental shift in microglial migratory behavior along vessels corresponded to when astrocyte endfeet more fully ensheath vessels. Together, our data provide a comprehensive assessment of microglia-vascular interactions. They support a mechanism by which microglia use the vasculature to migrate within the developing brain parenchyma. This migration becomes restricted on the arrival of astrocyte endfeet such that juxtavascular microglia become highly stationary and stable in the mature cortex.SIGNIFICANCE STATEMENT We report the first extensive analysis of juxtavascular microglia in the healthy, developing, and adult brain. Live imaging revealed that juxtavascular microglia within the cortex are highly motile and migrate along vessels as they are colonizing cortical regions. Using confocal, expansion, super-resolution, and electron microscopy, we determined that microglia associate with the vasculature at all ages in areas lacking full astrocyte endfoot coverage and motility of juxtavascular microglia ceases as astrocyte endfeet more fully ensheath the vasculature. Our data lay the fundamental groundwork to investigate microglia-astrocyte cross talk and juxtavascular microglial function in the healthy and diseased brain. They further provide a potential mechanism by which vascular interactions facilitate microglial colonization of the brain to later regulate neural circuit development.

Plasmalemmal Vesicle-Associated Protein Is Associated with Endothelial Cells Sprouting from the Peribiliary Capillary Plexus in Human Cirrhotic Liver.

INTRODUCTION: Plasmalemmal vesicle-associated protein (PLVAP) is an endothelial-specific integral membrane glycoprotein that localizes to caveolae and fenestrae in animal models; however, little is known about PLVAP in endothelial cells (ECs) in hepatic sinusoids during liver cirrhosis (LC). Here, we aimed to elucidate PLVAP localization and expression in the human liver during LC progression. METHODS: PLVAP protein expression was detected in specimens from normal control livers and hepatitis C-related cirrhotic livers using immunohistochemistry, Western blotting, and immunoelectron microscopy. RESULTS: PLVAP mainly localized to the peribiliary capillary plexus (PCP) and was rarely observed in hepatic artery branches and portal venules in control tissue, but was aberrantly expressed in capillarized sinusoids and proliferated capillaries in fibrotic septa within cirrhotic liver tissue. Ultrastructural analysis indicated that PLVAP localized to thin ECs in some caveolae, whereas PLVAP localized primarily to caveolae-like structures and proliferative sinusoid capillary EC vesicles in cirrhotic liver tissue. Western blot analysis confirmed that PLVAP was overexpressed at the protein level in advanced cirrhotic liver tissue. CONCLUSION: PLVAP was strongly expressed in the caveolae of proliferated capillaries directly connected with sinusoids linked with the PCP, suggesting that it plays a role in angiogenesis and sinusoidal remodeling in LC.

VDAC1 regulates mitophagy in NLRP3 inflammasome activation in retinal capillary endothelial cells under high-glucose conditions.

Diabetic retinopathy (DR) has been considered to involve mitochondrial alterations and be related to the nucleotide-binding oligomerization domain-like receptors 3 (NLRP3) inflammasome activation. The voltage-dependent anion channel 1 (VDAC1) protein is one of the key proteins that regulates the metabolic and energetic functions of the mitochondria. To explore the involvement of VDAC1 in mitophagy regulation of NLRP3 inflammasome activation under high-glucose (HG) conditions, this study examined expressions of VDAC1, mitochondrial function and mitophagy-related proteins, and NLRP3 inflammasome-related proteins in human retinal capillary endothelial cells (HRCECs) cultured with 30 mM of glucose in the presence or absence of mitophagy inhibitor (Mdivi-1) using Western blot. Mitochondrial membrane potential and mitochondrial reactive oxygen species (mtROS) were detected using flow cytometry. GFP-tagged pAdTrack-VDAC1 adenovirus was used to overexpress VDAC1. Cell biological behaviors, including proliferation, migration, tubule formation, and apoptosis, were also observed. Our results showed that when compared to the normal glucose and high mannitol groups, increased amounts of mitochondrial fragments, reduced mitochondrial membrane potential, increased expression of mitochondrial fission protein Drp 1, decreased expression of mitochondrial fusion protein Mfn 2, accumulation of mtROS, and activation of the NLRP3 inflammasome were observed in the HG group. Meanwhile, HG markedly reduced the protein expressions of PINK1, Parkin and VDAC1. Inhibition of mitophagy reduced PINK1 expression, enhanced NLRP3 expression, but failed to alter VDAC1. VDAC1 overexpression promoted PINK1 expression, inhibited NLRP3 activation and changed the cell biological behaviors under HG conditions. These findings demonstrate that VDAC1-mediated mitophagy plays a crucial role in regulating NLRP3 inflammasome activation in retinal capillary endothelial cells under HG conditions, suggesting that VDAC1 may be a potential target for preventing or treating DR.

Ultrastructure of Endothelial Cells of Myocardial Capillaries in Burn Septicotoxemia.

We studied ultrastructure and vesicular structures in endothelial cells of myocardial micro-vessels in burn patients. Electron microscopy revealed a significant decrease in volume density of vesicular structures in the endotheliocytes of myocardial capillaries in patients with burn septicotoxemia. The observed structural signs of endothelial dysfunction revealed in this category of patients can be a promising area for further research and for the development of methods of pathogenetic correction of myocardial disorders in the case of burn injury.

Consequences of being phenotypically mismatched with the environment: rapid muscle ultrastructural changes in cold-shocked black-capped chickadees (Poecile atricapillus).

Phenotypic flexibility has received considerable attention in the last decade; however, whereas many studies have reported amplitude of variation in phenotypic traits, much less attention has focused on the rate at which traits can adjust in response to sudden changes in the environment. We investigated whole animal and muscle phenotypic changes occurring in black-capped chickadees (Poecile atricapillus) acclimated to cold (-5°C) and warm (20°C) temperatures in the first 3 h following a 15°C temperature drop (over 3 h). Before the temperature change, cold-acclimated birds were consuming 95% more food, were carrying twice as much body fat, and had 23% larger pectoralis muscle fiber diameters than individuals kept at 20°C. In the 3 h following the temperature drop, these same birds altered their pectoralis muscle ultrastructure by increasing the number of capillaries per fiber area and the number of nuclei per millimeter of fiber by 22%, consequently leading to a 22% decrease in myonuclear domain (amount of cytoplasm serviced per nucleus), whereas no such changes were observed in the warm-acclimated birds. To our knowledge, this is the first demonstration of such a rapid adjustment in muscle fiber ultrastructure in vertebrates. These results support the hypothesis that chickadees maintaining a cold phenotype are better prepared than warm-phenotype individuals to respond to a sudden decline in temperature, such as what may be experienced in their natural wintering environment.

Selective induction of sprouting and intussusception is associated with the concentration distributions of oxygen and hypoxia-induced VEGF.

Sprouting and intussusception are two important modes of capillary angiogenesis, the mechanisms of selective induction of which remain unclear. In this study, we focus on the two developing tissues of yolk sac and skeletal muscle of 2-week-old rat and try to explain the mechanisms to induce selectively sprouting and intussusception in a new way to combine numerical calculation, experimental observations and schematic simulation. We propose the concept of capillary network unit and show that the concentration and gradient of oxygen/hypoxia-induced VEGF around straight segments are lower/higher than that around vascular bifurcations; sprouting mainly occurs at straight segments and intussusception at vascular bifurcations. The results indicate that the locations susceptible to sprouting and intussusception are determined by the distribution characteristics of oxygen/hypoxia-induced VEGF in the capillary network unit. Furthermore, it is considered that the flow dynamics at these locations also play important roles, namely laminar flow at straight segments promotes sprouting, and flow disruption at bifurcations promotes intussusception. Our work suggests the presence of the location preference for sprouting and intussusception, and provides a new research perspective to reveal its core mechanisms.

Sustained Ultrastructural Changes in Rat Hippocampal Formation After Repeated Electroconvulsive Seizures.

BACKGROUND: Electroconvulsive therapy (ECT) is a highly effective and fast-acting treatment for depression used in the clinic. Its mechanism of therapeutic action remains uncertain. Previous studies have focused on documenting neuroplasticity in the early phase following electroconvulsive seizures (ECS), an animal model of ECT. Here, we investigate whether changes in synaptic plasticity and nonneuronal plasticity (vascular and mitochondria) are sustained 3 months after repeated ECS trials. METHODS: ECS or sham treatment was given daily for 1 day or 10 days to a genetic animal model of depression: the Flinders Sensitive and Resistant Line rats. Stereological principles were employed to quantify numbers of synapses and mitochondria as well as length of microvessels in the hippocampus 24 hours after a single ECS. Three months after 10 ECS treatments (1 per day for 10 days) and sham-treatment, brain-derived neurotrophic factor and vascular endothelial growth factor protein levels were quantified with immunohistochemistry. RESULTS: A single ECS treatment significantly increased the volume of hippocampal CA1-stratum radiatum, the total length of microvessels, mitochondria number, and synapse number. Observed changes were sustained as shown in the multiple ECS treatment group analyzed 3 months after the last of 10 ECS treatments. CONCLUSION: A single ECS caused rapid effects of synaptic plasticity and nonneuronal plasticity, while repeated ECS induced long-lasting changes in the efficacy of synaptic plasticity and nonneuronal plasticity at least up to 3 months after ECS.

Notch-inducing hydrogels reveal a perivascular switch of mesenchymal stem cell fate.

The fate of mesenchymal stem cells (MSCs) in the perivascular niche, as well as factors controlling their fate, is poorly understood. Here, we study MSCs in the perivascular microenvironment of endothelial capillaries by modifying a synthetic 3D biomimetic poly(ethylene glycol) (PEG)-hydrogel system in vitro We show that MSCs together with endothelial cells form micro-capillary networks specifically in soft PEG hydrogels. Transcriptome analysis of human MSCs isolated from engineered capillaries shows a prominent switch in extracellular matrix (ECM) production. We demonstrate that the ECM phenotypic switch of MSCs can be recapitulated in the absence of endothelial cells by functionalizing PEG hydrogels with the Notch-activator Jagged1. Moreover, transient culture of MSCs in Notch-inducing microenvironments reveals the reversibility of this ECM switch. These findings provide insight into the perivascular commitment of MSCs by use of engineered niche-mimicking synthetic hydrogels.

Retinal and choroidal capillaries contribution to age-related macular degeneration (AMD) phenotypes in murine models of the disease.

Mouse models of age-related macular degeneration (AMD) such as Ccl2-/- and Ccl2-/-/Cx3cr1-/- have not yet been fully characterized ultrastructurally. Although we have previously shown extranuclear DNA (enDNA) leakage into the cytoplasm and damaged mitochondria in the retinal pigment epithelium (RPE) of these AMD mouse models, little is known about the state of their vascular capillaries of the retina and choroid. Our ultrastructural survey shows that the aberrations were not restricted to the RPE cells, but also extended to the vasculature of the retina and choroid. Their endothelial aberrations included cytoplasmic degeneration, pyknotic DNA, hypertrophic nuclei, and loss of fenestration in addition to duplication of basement membrane and loss of density in Bruch's membrane. Moreover, the state of the vasculature in the mutant mice models suggests that the capillaries could also be active contributors to the pathological findings seen in AMD. The goal of this study is to gain insights into the early events of AMD that may lead to a better understanding of AMD's pathogenesis, improve our preventative measures, and formulate designed therapeutic regimens that are tailored to target the initial pathological events.Abbreviations: AMD: age-related macular degeneration; BM: Bruch's membrane; DPC: degenerate pericyte; EN: endothelial nucleus; enDNA: extranuclear DNA; GCL: ganglion cell layer; HEN: hypertrophic endothelial nucleus; IPL: inner plexiform layer; NFL: nerve fiber layer; OPL: outer plexiform layer; RBC: red blood cell; RPE: retinal pigment epithelium; SNPs: Single nucleotide polymorphisms.

[MICRO- AND ULTRASTRUCTURAL RECONSTRUCTION OF THE RAT SUBMANDIBULAR GLAND IN THE LATE STAGES OF EXPERIMENTAL TYPE 2 DIABETES MELLITUS].

Electron microscopic investigations of the animals' submandibular gland, conducted in 6 weeks of the experiment, established that ultrastructural changes increase in glandular cells of terminal secretory units in comparison with early period of the experiment. Serocytes have osmiophilic, rather small or picnotic nuclei. Perinuclear spaces of karyolemma are uneven, external nuclear membrane forms local protrusions. Electron density of the karyoplasm is significant, appearing homogenous, nuclei are not observed. Evident submicroscopic changes in blood capillaries of the submandibular gland in experimental diabetes mellitus indicate the impairment of blood-tissue barrier and transcapillary exchange. Deep destructive modifications of all branches of microcirculatory blood flow of the submandibular gland are observed in 8-week course of experimental diabetes mellitus. As compared with the 6th week of investigation, a reliable slight dilation of organ artery diameter, dilation of the diameter of interlobular arterioles, dilation of the diameter of intralobular (precapillary) arteriole and dilation of the capillary diameter were observed. Dilation, as compared to 6th weeks of the experiment, of postcapillary venules was observed. Compared to the indices of the 6th weeks of the experiment, an index of trophic activity of the submandibular gland tissuereaches its maximum meaning and an index of packing density of the capillaries reaches its minimum meaning. Capillary network loses delicate, tortuous pattern and often breaks due to destruction of the capillary component. Arteriovenous anastomoses dilate and blood from the arterioles flows into the venous bed avoiding destructed capillaries. Venules are dilated; thin-walled, retained fragments of the capillaries are significantly dilated in some areas. Swelling of connective tissue stroma and significant swelling of the interstitium are observed. Walls of the capillaries and venules are deformed. The walls of the arterioles are thickened due to plasmorrhagia, sclerosis and hyalinosis.

Age-related ultrastructural changes of the basement membrane in the mouse blood-brain barrier.

The blood-brain barrier (BBB) is essential for a functional neurovascular unit. Most studies focused on the cells forming the BBB, but very few studied the basement membrane (BM) of brain capillaries in ageing. We used transmission electron microscopy and electron tomography to investigate the BM of the BBB in ageing C57BL/6J mice. The thickness of the BM of the BBB from 24-month-old mice was double as compared with that of 6-month-old mice (107 nm vs 56 nm). The aged BBB showed lipid droplets gathering within the BM which further increased its thickness (up to 572 nm) and altered its structure. The lipids appeared to accumulate toward the glial side of the BM. Electron tomography showed that the lipid-rich BM regions are located in small pockets formed by the end-feet of astrocytes. These findings suggest an imbalance of the lipid metabolism and that may precede the structural alteration of the BM. These alterations may favour the accretion of abnormal proteins that lead to neurodegeneration in ageing. These findings warrant further investigation of the BM of brain capillaries and of adjoining cells as potential targets for future therapies.

Endothelin receptor-A mediates degradation of the glomerular endothelial surface layer via pathologic crosstalk between activated podocytes and glomerular endothelial cells.

Emerging evidence of crosstalk between glomerular cells in pathological settings provides opportunities for novel therapeutic discovery. Here we investigated underlying mechanisms of early events leading to filtration barrier defects of podocyte and glomerular endothelial cell crosstalk in the mouse models of primary podocytopathy (podocyte specific transforming growth factor-ß receptor 1 signaling activation) or Adriamycin nephropathy. We found that glomerular endothelial surface layer degradation and albuminuria preceded podocyte foot process effacement. These abnormalities were prevented by endothelin receptor-A antagonism and mitochondrial reactive oxygen species scavenging. Additional studies confirmed increased heparanase and hyaluronoglucosaminidase gene expression in glomerular endothelial cells in response to podocyte-released factors and to endothelin-1. Atomic force microscopy measurements showed a significant reduction in the endothelial surface layer by endothelin-1 and podocyte-released factors, which could be prevented by endothelin receptor-A but not endothelin receptor-B antagonism. Thus, our studies provide evidence of early crosstalk between activated podocytes and glomerular endothelial cells resulting in loss of endothelial surface layer, glomerular endothelial cell injury and albuminuria. Hence, activation of endothelin-1-endothelin receptor-A and mitochondrial reactive oxygen species contribute to the pathogenesis of primary podocytopathies in experimental focal segmental glomerulosclerosis.

High thoracic sympathetic block improves coronary microcirculation disturbance in rats with chronic heart failure.

INTRODUCTION: Coronary microcirculation disturbance plays an important role in chronic heart failure (CHF). High thoracic sympathetic block (HTSB) is effective to treat CHF, but its impact on coronary microcirculation is unclear. METHODS: Forty male Wistar rats were subcutaneously injected with isoproterenol (340 mg/kg) for 2 days. Eight weeks later, 24 surviving rats were randomized to the CHF and HTSB groups and 10 rats were used as the control group. 50 µl of saline and ropivacaine (0.2%) were epidurally infused everyday in the CHF and HTSB group respectively. Four weeks later, echocardiography and pathological and ultrastructural examination, capillary histochemical staining and vascular endothelial growth factor (VEGF) immunohistochemical staining in left ventricular (LV) subendocardial myocardium were performed. RESULTS: Compared with the control group, LV dilation and dysfunction, myocardial focal necrosis, capillary spasm appeared in the CHF group. HTSB ameliorated LV dilation and dysfunction, myocardial necrosis and capillary spasm. Rats in the CHF group had less myocardial capillary density and more VEGF expression than in the control group (1591 ±â€¯99 vs. 1972 ±â€¯118/mm2, 0.62 ±â€¯0.13 vs. 0.33 ±â€¯0.10 optic density, all p < 0.05). Myocardial capillary density (1782 ±â€¯96/mm2) was more and VEGF expression (0.47 ±â€¯0.12 optic density) was less in the HTSB group than in the CHF group (all p < 0.05). CONCLUSION: HTSB improves coronary microcirculation disturbance in CHF, which may contribute to reversing myocardial remodeling and dysfunction. HTSB stimulates myocardial capillary growth independent of VEGF.

Lamina propria: The connective tissue of rat urinary bladder mucosa.

To describe and illustrate the structure of the propria, the bladder of adult rats was fixed in controlled conditions of distension and examined by light and electron microscopy. The lamina propria, ~50 µm thick in the distended bladder, consists of a superficial part (the cellular component), adjacent to the urothelium, rich in nerves, capillaries, fibroblasts and thin bundles of collagen, and a deep, thicker part (the fibrous component), adjacent to the detrusor, rich in large collagen fibres and with few fibroblasts. In the cellular part there is an extensive plexus of afferent nerve fibers and a dense capillary network (with numerous pericytes), lying close to the urothelium, that is unique to the bladder. The main resident cells are fibroblasts, adhering to each other at the end of laminar extensions without forming specialized junctions. The deep part of the lamina propria is made of thick collagen fibers, interwoven and crisscrossing each other, with a few fibroblasts in the interstitial spaces between them. In summary, the superficial part of the lamina propria has most of the bladder afferent nerves, contains many fibroblasts and has a network of suburothelial capillaries. The deep part as a whole forms an ovoid balloon of woven fibrous material that is acted upon by the detrusor musculature attached to its outer surface. The lamina propria is a strong fibrous barrier between urothelium and musculature. The abundance of collagen points to the main role for its fibroblasts, that is, the production of collagen fibrils, assisting the mechanical role of the lamina propria.

The effect of two exercise modalities on skeletal muscle capillary ultrastructure in individuals with type 2 diabetes.

Type 2 diabetes is associated with microvascular dysfunction, but little is known about how capillary ultrastructure is affected by exercise training. To investigate the effect of two types of exercise training on skeletal muscle capillary ultrastructure and capillarization in individuals with type 2 diabetes, 21 individuals with type 2 diabetes were allocated (randomized controlled trial) to 11 weeks of aerobic exercise training consisting of either moderate-intensity endurance training (END; n = 10) or low-volume high-intensity interval training (HIIT; n = 11). Skeletal muscle biopsies (m vastus lateralis) were obtained before and after the training intervention. At baseline, there was no difference in capillarization, capillary structure, and exercise hyperemia between the two groups. After the training intervention, capillary-to-fiber ratio increased by 8% ± 3% in the END group (P < 0.05) and was unchanged in the HIIT group with no difference between groups. Endothelium thickness increased (P < 0.05), basement membrane thickness decreased (P < 0.05), and the capillary lumen tended (P = 0.07) to increase in the END group, whereas these structural indicators were unchanged after HIIT. In contrast, skeletal muscle endothelial nitric oxide synthase (eNOS) increased after HIIT (P < 0.05), but not END, whereas there was no change in vascular endothelial growth factor (VEGF), superoxide dismutase (SOD)-2, or NADPH oxidase after both training protocols. In contrast to END training, HIIT did not alter capillarization or capillary structure in individuals with type 2 diabetes. In conclusion, HIIT appears to be a less effective strategy to treat capillary rarefaction and reduce basement thickening in type 2 diabetes.

Characterization of isolated liver sinusoidal endothelial cells for liver bioengineering.

BACKGROUND: The liver sinusoidal capillaries play a pivotal role in liver regeneration, suggesting they may be beneficial in liver bioengineering. This study isolated mouse liver sinusoidal endothelial cells (LSECs) and determined their ability to form capillary networks in vitro and in vivo for liver tissue engineering purposes. METHODS AND RESULTS: In vitro LSECs were isolated from adult C57BL/6 mouse livers. Immunofluorescence labelling indicated they were LYVE-1+/CD32b+/FactorVIII+/CD31-. Scanning electron microscopy of LSECs revealed the presence of characteristic sieve plates at 2 days. LSECs formed tubes and sprouts in the tubulogenesis assay, similar to human microvascular endothelial cells (HMEC); and formed capillaries with lumens when implanted in a porous collagen scaffold in vitro. LSECs were able to form spheroids, and in the spheroid gel sandwich assay produced significantly increased numbers (p = 0.0011) of capillary-like sprouts at 24 h compared to HMEC spheroids. Supernatant from LSEC spheroids demonstrated significantly greater levels of vascular endothelial growth factor-A and C (VEGF-A, VEGF-C) and hepatocyte growth factor (HGF) compared to LSEC monolayers (p = 0.0167; p = 0.0017; and p < 0.0001, respectively), at 2 days, which was maintained to 4 days for HGF (p = 0.0017) and VEGF-A (p = 0.0051). In vivo isolated mouse LSECs were prepared as single cell suspensions of 500,000 cells, or as spheroids of 5000 cells (100 spheroids) and implanted in SCID mouse bilateral vascularized tissue engineering chambers for 2 weeks. Immunohistochemistry identified implanted LSECs forming LYVE-1+/CD31- vessels. In LSEC implanted constructs, overall lymphatic vessel growth was increased (not significantly), whilst host-derived CD31+ blood vessel growth increased significantly (p = 0.0127) compared to non-implanted controls. LSEC labelled with the fluorescent tag DiI prior to implantation formed capillaries in vivo and maintained LYVE-1 and CD32b markers to 2 weeks. CONCLUSION: Isolated mouse LSECs express a panel of vascular-related cell markers and demonstrate substantial vascular capillary-forming ability in vitro and in vivo. Their production of liver growth factors VEGF-A, VEGF-C and HGF enable these cells to exert a growth stimulus post-transplantation on the in vivo host-derived capillary bed, reinforcing their pro-regenerative capabilities for liver tissue engineering studies.

Intussusceptive Pillar Formation in Developing Porcine Glomeruli.

BACKGROUND/AIMS: Intussusceptive angiogenesis (IA) is a dynamic process which contributes to vascular expansion and remodeling. Intraluminal pillars have long been the distinctive structural indicator of IA. However, the mechanism of their formation has not been fully elucidated. METHODS: Using light and electron microscopy, we studied intussusceptive vascular growth in the developing porcine metanephric kidney. RESULTS: We observed intraluminal pillars formed by endothelial cells in the vasculature of developing glomeruli. Their diameter was < 2.5 µm, consistent with the diameter of nascent pillars. TEM revealed that the majority of these pillars consisted only of endothelium. However, a central core of extracellular matrix (ECM) covered by endothelium, reminiscent of a more mature intussusceptive pillar, was also found in the lumen of a glomerular capillary. Perivascular cells or pericytes were not involved in the pillar structure during these stages of formation. CONCLUSION: This study shows ECM presence in a mature intussusceptive pillar without any perivascular cell involvement in the structure. This leads to the hypothesis that ECM deposition precedes the participation of these cells in the formation of intraluminal pillars during IA in porcine metanephric glomerular capillaries.

Intravenously Transplanted Human Bone Marrow Endothelial Progenitor Cells Engraft Within Brain Capillaries, Preserve Mitochondrial Morphology, and Display Pinocytotic Activity Toward Blood-Brain Barrier Repair in Ischemic Stroke Rats.

Stroke is a life-threatening disease with limited therapeutic options. Cell therapy has emerged as an experimental stroke treatment. Blood-brain barrier (BBB) impairment is a key pathological manifestation of ischemic stroke, and barrier repair is an innovative target for neurorestoration in stroke. Here, we evaluated via electron microscopy the ability of transplanted human bone marrow endothelial progenitor cells (hBMEPCs) to repair the BBB in adult Sprague-Dawley rats subjected to transient middle cerebral artery occlusion (tMCAO). ß-galactosidase prelabeled hBMEPCs were intravenously transplanted 48 hours post-tMCAO. Ultrastructural analysis of microvessels in nontransplant stroke rats revealed typical BBB pathology. At 5 days post-transplantation with hBMEPCs, stroke rats displayed widespread vascular repair in bilateral striatum and motor cortex, characterized by robust cell engraftment within capillaries. hBMEPC transplanted stroke rats exhibited near normal morphology of endothelial cells (ECs), pericytes, and astrocytes, without detectable perivascular edema. Near normal morphology of mitochondria was also detected in ECs and perivascular astrocytes from transplanted stroke rats. Equally notable, we observed numerous pinocytic vesicles within engrafted cells. Robust engraftment and intricate functionality of transplanted hBMEPCs likely abrogated stroke-altered vasculature. Preserving mitochondria and augmenting pinocytosis in cell-based therapeutics represent a new neurorestorative mechanism in BBB repair for stroke. Stem Cells 2017;35:1246-1258.

An unusual pattern of peritubular capillary injury involving apoptosis in a renal transplant patient.

Microvascular injury is an important factor in renal allograft survival. Repeated episodes of endothelial injury from chronic antibody-mediated rejection typically manifest at the ultrastructural level as circumferential multilayering of remodeled glomerular basement membrane material and peritubular capillary basal lamina. In contrast to this typical pattern of microvascular injury, a renal transplantation case is presented in which focally dilated and multilayered segments of peritubular capillary basal lamina bearing lipid droplets were interspersed with ultrastructurally normal unilayered segments of basal lamina devoid of lipid droplets. Glomerular basement membranes were not affected by this process. The peak incidence of lipid droplets within the peritubular capillary walls coincided with a peak in apoptotic activity within the allograft. Lesser amounts of the same lipidic material were identified in the mesangial matrix and an arteriolar wall. Mesangial electron-dense deposits were detected at two weeks posttransplantation and their appearance coincided with elevated immunological activity in the glomeruli, as determined by immunofluorescence microscopy. The unusual ultrastructure and immunological activity observed in this case may reflect a process of impaired apoptotic clearance within the allograft. The six biopsies from a single patient are discussed in the setting of a highly sensitized renal transplant recipient who received prophylactic terminal complement blockade by eculizumab. The findings may be relevant to the study of apoptosis, efferocytosis, microvascular injury, eculizumab, rejection, lupus, and drug-related disease.