Liver Environment-Imposed Constraints Diversify Movement Strategies of Liver-Localized CD8 T Cells.

Pathogen-specific CD8 T cells face the problem of finding rare cells that present their cognate Ag either in the lymph node or in infected tissue. Although quantitative details of T cell movement strategies in some tissues such as lymph nodes or skin have been relatively well characterized, we still lack quantitative understanding of T cell movement in many other important tissues, such as the spleen, lung, liver, and gut. We developed a protocol to generate stable numbers of liver-located CD8 T cells, used intravital microscopy to record movement patterns of CD8 T cells in livers of live mice, and analyzed these and previously published data using well-established statistical and computational methods. We show that, in most of our experiments, Plasmodium-specific liver-localized CD8 T cells perform correlated random walks characterized by transiently superdiffusive displacement with persistence times of 10-15 min that exceed those observed for T cells in lymph nodes. Liver-localized CD8 T cells typically crawl on the luminal side of liver sinusoids (i.e., are in the blood); simulating T cell movement in digital structures derived from the liver sinusoids illustrates that liver structure alone is sufficient to explain the relatively long superdiffusive displacement of T cells. In experiments when CD8 T cells in the liver poorly attach to the sinusoids (e.g., 1 wk after immunization with radiation-attenuated Plasmodium sporozoites), T cells also undergo Lévy flights: large displacements occurring due to cells detaching from the endothelium, floating with the blood flow, and reattaching at another location. Our analysis thus provides quantitative details of movement patterns of liver-localized CD8 T cells and illustrates how structural and physiological details of the tissue may impact T cell movement patterns.

Microfluidic device engineered to study the trafficking of multiple myeloma cancer cells through the sinusoidal niche of bone marrow.

Multiple myeloma (MM) is an incurable B cell malignancy characterized by the accumulation of monoclonal abnormal plasma cells in the bone marrow (BM). It has been a significant challenge to study the spatiotemporal interactions of MM cancer cells with the embedded microenvironments of BM. Here we report a microfluidic device which was designed to mimic several physiological features of the BM niche: (1) sinusoidal circulation, (2) sinusoidal endothelium, and (3) stroma. The endothelial and stromal compartments were constructed and used to demonstrate the device's utility by spatiotemporally characterizing the CXCL12-mediated egression of MM cells from the BM stroma and its effects on the barrier function of endothelial cells (ECs). We found that the egression of MM cells resulted in less organized and loosely connected ECs, the widening of EC junction pores, and increased permeability through ECs, but without significantly affecting the number density of viable ECs. The results suggest that the device can be used to study the physical and secreted factors determining the trafficking of cancer cells through BM. The sinusoidal flow feature of the device provides an integral element for further creating systemic models of cancers that reside or metastasize to the BM niche.

Microgravity and space radiation inhibit autophagy in human capillary endothelial cells, through either opposite or synergistic effects on specific molecular pathways.

Microgravity and space radiation (SR) are two highly influential factors affecting humans in space flight (SF). Many health problems reported by astronauts derive from endothelial dysfunction and impaired homeostasis. Here, we describe the adaptive response of human, capillary endothelial cells to SF. Reference samples on the ground and at 1g onboard permitted discrimination between the contribution of microgravity and SR within the combined responses to SF. Cell softening and reduced motility occurred in SF cells, with a loss of actin stress fibers and a broader distribution of microtubules and intermediate filaments within the cytoplasm than in control cells. Furthermore, in space the number of primary cilia per cell increased and DNA repair mechanisms were found to be activated. Transcriptomics revealed the opposing effects of microgravity from SR for specific molecular pathways: SR, unlike microgravity, stimulated pathways for endothelial activation, such as hypoxia and inflammation, DNA repair and apoptosis, inhibiting autophagic flux and promoting an aged-like phenotype. Conversely, microgravity, unlike SR, activated pathways for metabolism and a pro-proliferative phenotype. Therefore, we suggest microgravity and SR should be considered separately to tailor effective countermeasures to protect astronauts' health.

Bone marrow sinusoidal endothelium controls terminal erythroid differentiation and reticulocyte maturation.

Within the bone marrow microenvironment, endothelial cells (EC) exert important functions. Arterial EC support hematopoiesis while H-type capillaries induce bone formation. Here, we show that BM sinusoidal EC (BM-SEC) actively control erythropoiesis. Mice with stabilized ß-catenin in BM-SEC (Ctnnb1OE-SEC) generated by using a BM-SEC-restricted Cre mouse line (Stab2-iCreF3) develop fatal anemia. While activation of Wnt-signaling in BM-SEC causes an increase in erythroblast subsets (PII-PIV), mature erythroid cells (PV) are reduced indicating impairment of terminal erythroid differentiation/reticulocyte maturation. Transplantation of Ctnnb1OE-SEC hematopoietic stem cells into wildtype recipients confirms lethal anemia to be caused by cell-extrinsic, endothelial-mediated effects. Ctnnb1OE-SEC BM-SEC reveal aberrant sinusoidal differentiation with altered EC gene expression and perisinusoidal ECM deposition and angiocrine dysregulation with de novo endothelial expression of FGF23 and DKK2, elevated in anemia and involved in vascular stabilization, respectively. Our study demonstrates that BM-SEC play an important role in the bone marrow microenvironment in health and disease.

Loss with ageing but preservation of frontal cortical capillary pericytes in post-stroke dementia, vascular dementia and Alzheimer's disease.

Cerebral pericytes are an integral component of the neurovascular unit, which governs the blood-brain barrier. There is paucity of knowledge on cortical pericytes across different dementias. We quantified cortical pericytes in capillaries in 124 post-mortem brains from subjects with post-stroke dementia (PSD), vascular dementia (VaD), Alzheimer's disease (AD) and AD-VaD (Mixed) and, post-stroke non-demented (PSND) stroke survivors as well as normal ageing controls. Collagen 4 (COL4)-positive nucleated pericyte soma were identified as protrusions on capillaries of the frontal cortex. The COL4-positive somata or nodule-like cell bodies were also verified by platelet derived growth factor receptor-ß (PDGFR-ß) immunohistochemistry. The mean (± SEM) pericyte somata in frontal cortical capillaries in normal young controls (46-65 years of age) was estimated as 5.2 ± 0.2 per mm capillary length. This number was reduced by 45% in older controls (> 78 years) to 2.9 ± 0.1 per mm capillary length (P < 0.001). We further found that the numbers of pericyte cell bodies per COL4 mm2 area or per mm capillary length were not decreased but rather preserved or increased in PSD, AD and Mixed dementia groups compared to similar age older controls (P < 0.01). Consistent with this, we noted that capillary length densities identified by the endothelial marker glucose transporter 1 or COL4 were not different across the dementias compared to older controls. There was a negative correlation with age (P < 0.001) suggesting fewer pericyte somata in older age, although the % COL4 immunoreactive capillary area was increased in older controls compared to young controls. Using a proven reliable method to quantify COL4-positive nucleated pericytes, our observations demonstrate ageing related loss but mostly preserved pericytes in the frontal cortex of vascular and AD dementias. We suggest there is differential regulation of capillary pericytes in the frontal lobe between the cortex and white matter in ageing-related dementias.

Distributions of Radial Peripapillary Capillary Density and Correlations with Retinal Nerve Fiber Layer Thickness in Normal Subjects.

BACKGROUND The aim of this study was to investigate distribution rules of radial peripapillary capillaries (RPCs) density and correlations with retinal nerve fiber layers (RNFL) thickness in normal subjects. MATERIAL AND METHODS We included 78 eyes of 78 healthy subjects examined by optical coherence tomography angiography (OCTA). RPCs density and RNFL thickness were measured automatically. Distributions of RPCs density and RNFL thickness were analyzed at different locations. Correlations of these 2 parameters and relationship with large vessels were evaluated by Spearman test. RESULTS Average density for overall, peripapillary, and inside disc RCPs was 56.12±2.51%, 58.56±2.84%, and 60.16±4.01%, respectively. Overall and peripapillary RCPs density were positively correlated with RNFL thickness (r=0.595, P.

Point-of-care microvolume cytometer measures platelet counts with high accuracy from capillary blood.

BACKGROUND: Point-of-care (PoC) testing of platelet count (PLT) provides real-time data for rapid decision making. The goal of this study is to evaluate the accuracy and precision of platelet counting using a new microvolume (8 µL), absolute counting, 1.5 kg cytometry-based blood analyzer, the rHEALTH ONE (rHEALTH) in comparison with the International Society of Laboratory Hematology (ISLH) platelet method, which uses a cytometer and an impedance analyzer. METHODS: Inclusion eligibility were healthy adults (M/F) ages 18-80 for donation of fingerprick and venous blood samples. Samples were from a random N = 31 volunteers from a single U.S. site. Samples were serially diluted to test thrombocytopenic ranges. Interfering substances and conditions were tested, including RBC fragments, platelet fragments, cholesterol, triglycerides, lipids, anti-platelet antibodies, and temperature. RESULTS: The concordance between the rHEALTH and ISLH methods had a slope = 1.030 and R2 = 0.9684. The rHEALTH method showed a correlation between capillary and venous blood samples (slope = 0.9514 and R2 = 0.9684). Certain interferents changed platelet recovery: RBC fragments and anti-platelet antibodies with the ISLH method; platelet fragments and anti-platelet antibodies on the rHEALTH; and RBC fragments, platelets fragments, triglycerides and LDL on the clinical impedance analyzer. The rHEALTH's precision ranged from 3.1-8.0%, and the ISLH from 1.0-10.5%. CONCLUSIONS: The rHEALTH method provides similar results with the reference method and good correlation between adult capillary and venous blood samples. This demonstrates the ability of the rHEALTH to provide point-of-care assessment of normal and thrombocytopenic platelet counts from fingerprick blood with high precision and limited interferences.

HepaticIschemia/Reperfusion Injuryinvolves functional tryptase/PAR-2 signaling in liver sinusoidal endothelial cell population.

Mast cells (MCs) are tissue-resident effector cells that could be the earliest responder to release a unique, stimulus-specific set of mediators in hepatic ischemia-reperfusion (IR) injury However, how MCs function in the hepatic IR has remained a formidable challenge due to the substantial redundancy and functional diverse of these mediators. Tryptase is the main protease for degranulation of MCs and its receptor-protease-activated receptor 2 (PAR-2) is widely expressed in endothelial cells. It is unclear whether and how tryptase/PAR-2 axis participates in hepatic IR. We employed an experimental warm 70% liver IR model in mice and found that tryptase was accumulated in the circulation during hepatic IR and positively correlated with liver injury. Tryptase inhibition by protamine can significantly down-regulate the expression of adhesion molecules and reduce neutrophil infiltration within the liver. The level of inflammatory factors and chemokines were also consistent with the pathological change of the liver. In addition, the treatment with exogeneous tryptase in MC-deficient mice can induce the damage observed in wild type mice in the context of liver IR. In vitro, neutrophil infiltration and inflammatory factor secretion were regulated by Tryptase/PAR-2, involving the adhesion molecule expression to regulate neutrophil adhesion dependent on NF-κB pathway. Conclusion: tryptase/PAR-2 participates in liver injury through the activation of LSECs in the early phase of liver IR.

Techniques used to assess intussusceptive angiogenesis: A systematic review.

Intussusceptive angiogenesis (IA) is an important physiological form of angiogenesis in which an existing vessel splits in two by the formation of an intraluminal tissue pillar. The presence of these intraluminal pillars form the hallmark of ongoing IA in growing vascular beds. However, their visualization is technically challenging. The goal of this systematic review was to investigate which techniques are being used to identify intraluminal pillars and to formulate important points to keep in mind when studying IA. A systematic literature search resulted in 154 evaluated articles of which the majority (65%) provided sufficient data to unambiguously demonstrate the presence of intraluminal pillars. Scanning electron microscopy imaging of vascular corrosion casts and serial sectioning of ultrathin sections are the most used techniques. New methods such as serial block face scanning electron microscopy and micro computed tomography (µCT) are gaining importance. Moreover, our results indicate that IA was studied in a variety of animals and tissues. IA is a biologically very relevant form of angiogenesis. Techniques to visualize intraluminal pillars need to have a minimal resolution of 1 µm and should provide information on the 3D-nature of the pillars. Optimally, several techniques are combined to demonstrate ongoing IA.

Single-cell transcriptome profiling reveals vascular endothelial cell heterogeneity in human skin.

Vascular endothelial cells (ECs) are increasingly recognized as active players in intercellular crosstalk more than passive linings of a conduit for nutrition delivery. Yet, their functional roles and heterogeneity in skin remain uncharacterized. We have used single-cell RNA sequencing (scRNA-seq) as a profiling strategy to investigate the tissue-specific features and intra-tissue heterogeneity in dermal ECs at single-cell level. Methods: Skin tissues collected from 10 donors were subjected to scRNA-seq. Human dermal EC atlas of over 23,000 single-cell transcriptomes was obtained and further analyzed. Arteriovenous markers discovered in scRNA-seq were validated in human skin samples via immunofluorescence. To illustrate tissue-specific characteristics of dermal ECs, ECs from other human tissues were extracted from previously reported data and compared with our transcriptomic data. Results: In comparison with ECs from other human tissues, dermal ECs possess unique characteristics in metabolism, cytokine signaling, chemotaxis, and cell adhesions. Within dermal ECs, 5 major subtypes were identified, which varied in molecular signatures and biological activities. Metabolic transcriptome analysis revealed a preference for oxidative phosphorylation in arteriole ECs when compared to capillary and venule ECs. Capillary ECs abundantly expressed HLA-II molecules, suggesting its immune-surveillance role. Post-capillary venule ECs, with high levels of adhesion molecules, were equipped with the capacity in immune cell arrest, adhesion, and infiltration. Conclusion: Our study provides a comprehensive characterization of EC features and heterogeneity in human dermis and sets the stage for future research in identifying disease-specific alterations of dermal ECs in various dermatoses.

The frequency window effect of sinusoidal electromagnetic fields in promoting osteogenic differentiation and bone formation involves extension of osteoblastic primary cilia and activation of protein kinase A.

Electromagnetic fields (EMFs) have emerged as a versatile means for osteoporosis treatment and prevention. However, its optimal application parameters are still elusive. Here, we optimized the frequency parameter first by cell culture screening and then by animal experiment validation. Osteoblasts isolated from newborn rats (ROBs) were exposed 90 min/day to 1.8 mT SEMFs at different frequencies (ranging from 10 to 100 Hz, interval of 10 Hz). SEMFs of 1.8 mT inhibited ROB proliferation at 30, 40, 50, 60 Hz, but increased proliferation at 10, 70, 80 Hz. SEMFs of 10, 50, and 70 Hz promoted ROB osteogenic differentiation and mineralization as shown by alkaline phosphatase (ALP) activity, calcium content, and osteogenesis-related molecule expression analyses, with 50 Hz showing greater effects than 10 and 70 Hz. Treatment of young rats with 1.8 mT SEMFs at 10, 50, or 100 Hz for 2 months significantly increased whole-body bone mineral density (BMD) and femur microarchitecture, with the 50 Hz group showing the greatest effect. Furthermore, 1.8 mT SEMFs extended primary cilia lengths of ROBs and increased protein kinase A (PKA) activation also in a frequency-dependent manner, again with 50 Hz SEMFs showing the greatest effect. Pretreatment of ROBs with the PKA inhibitor KT5720 abolished the effects of SEMFs to increase primary cilia length and promote osteogenic differentiation/mineralization. These results indicate that 1.8 mT SEMFs have a frequency window effect in promoting osteogenic differentiation/mineralization in ROBs and bone formation in growing rats, which involve osteoblast primary cilia length extension and PKA activation.

Oxidized low-density lipoprotein activates extracellular signal-regulated kinase signaling to downregulate sortilin expression in liver sinusoidal endothelial cells.

BACKGROUND AND AIM: Both type 2 diabetes mellitus and non-alcoholic fatty liver disease are closely associated with elevated levels of low-density lipoprotein cholesterol and its oxidized form (ox-LDL). This study aimed to investigate the regulation of sortilin in liver tissue and its potential implications for lipid metabolism. METHODS: Sixty male Wistar rats were randomly divided into four groups: control group (n = 15), ox-LDL group (n = 15), PD98059 group (n = 15), and ox-LDL + PD98059 group (n = 15). Liver sinusoidal endothelial cells were extracted from liver tissue of the control group and were identified using an anti-CD31 antibody. Lipid droplet accumulation was observed by Oil red O and hematoxylin-eosin staining. The protein expression levels were detected by immunohistochemical staining, real-time reverse transcription-polymerase chain reaction, and western blot. Histopathologic examinations were performed by Gomori methenamine silver staining. RESULTS: The ox-LDL group exhibited increased lipid droplet accumulation. Further, ox-LDL activated the extracellular signal-regulated kinase (ERK)-mediated downregulation of sortilin expression, whereas blocking of ERK signaling by PD98059 increased sortilin protein expression. Consistently, hematoxylin-eosin staining showed that the structure of the hepatocytes was loose and disordered in arrangement, with lipid droplets present in the cytoplasm of the ox-LDL group. However, PD98059 significantly improved the integration of the scaffold structure. Gomori methenamine silver staining showed that the ox-LDL group had darker and more obvious fragmented silver nitrate deposits in the basement membrane and sinus space. CONCLUSIONS: Sortilin can protect liver sinusoidal endothelial cells from injury and maintain integration of the liver scaffold structure in ox-LDL-induced lipid-injured liver.

Capabilities of selenoneine to cross the in vitro blood-brain barrier model.

The naturally occurring selenoneine (SeN), the selenium analogue of the sulfur-containing antioxidant ergothioneine, can be found in high abundance in several marine fish species. However, data on biological properties of SeN and its relevance for human health are still scarce. This study aims to investigate the transfer and presystemic metabolism of SeN in a well-established in vitro model of the blood-brain barrier (BBB). Therefore, SeN and the reference Se species selenite and Se-methylselenocysteine (MeSeCys) were applied to primary porcine brain capillary endothelial cells (PBCECs). Se content of culture media and cell lysates was measured via ICP-MS/MS. Speciation analysis was conducted by HPLC-ICP-MS. Barrier integrity was shown to be unaffected during transfer experiments. SeN demonstrated the lowest transfer rates and permeability coefficient (6.7 × 10-7 cm s-1) in comparison to selenite and MeSeCys. No side-directed accumulation was observed after both-sided application of SeN. However, concentration-dependent transfer of SeN indicated possible presence of transporters on both sides of the barrier. Speciation analysis demonstrated no methylation of SeN by the PBCECs. Several derivatives of SeN detected in the media of the BBB model were also found in cell-free media containing SeN and hence not considered to be true metabolites of the PBCECs. In concluding, SeN is likely to have a slow transfer rate to the brain and not being metabolized by the brain endothelial cells. Since this study demonstrates that SeN may reach the brain tissue, further studies are needed to investigate possible health-promoting effects of SeN in humans.

Accelerated Clearance and Degradation of Cell-Free HIV by Neutralizing Antibodies Occurs via FcγRIIb on Liver Sinusoidal Endothelial Cells by Endocytosis.

Neutralizing Abs suppress HIV infection by accelerating viral clearance from blood circulation in addition to neutralization. The elimination mechanism is largely unknown. We determined that human liver sinusoidal endothelial cells (LSEC) express FcγRIIb as the lone Fcγ receptor, and using humanized FcγRIIb mouse, we found that Ab-opsonized HIV pseudoviruses were cleared considerably faster from circulation than HIV by LSEC FcγRIIb. Compared with humanized FcγRIIb-expressing mice, HIV clearance was significantly slower in FcγRIIb knockout mice. Interestingly, a pentamix of neutralizing Abs cleared HIV faster compared with hyperimmune anti-HIV Ig (HIVIG), although the HIV Ab/Ag ratio was higher in immune complexes made of HIVIG and HIV than pentamix and HIV. The effector mechanism of LSEC FcγRIIb was identified to be endocytosis. Once endocytosed, both Ab-opsonized HIV pseudoviruses and HIV localized to lysosomes. This suggests that clearance of HIV, endocytosis, and lysosomal trafficking within LSEC occur sequentially and that the clearance rate may influence downstream events. Most importantly, we have identified LSEC FcγRIIb-mediated endocytosis to be the Fc effector mechanism to eliminate cell-free HIV by Abs, which could inform development of HIV vaccine and Ab therapy.

Assessment of Pericyte Phenotype by Flow Cytometry.

Pericytes are mural cells closely associated with endothelial cells in capillaries and microvessels. They are precursors of mesenchymal stem/stromal cells that have historically been retrospectively characterized in culture. We established a protocol, described in this chapter, to characterize and isolate pericytes from multiple human organs by flow cytometry and fluorescence-activated cell sorting. This prospective purification of pericytes brings us a step forward in the development of strategies for their use in the clinic.

Characterization of Human Pericyte Phenotype by Immunohistochemistry.

Pericytes are found in all vascularized organs and are defined anatomically as perivascular cells that closely surround endothelial cells in capillaries and microvessels and are embedded within the same basement membrane. They have been shown to have diverse physiological and pathological functions including regulation of blood pressure, and tissue regeneration and scarring. Fundamental to understanding the role these cells play in these diverse processes is the ability to accurately identify and localize them in vivo. To do this, we have developed multicolor immunohistochemistry protocols described in this chapter.

A Metabolomics-Based Approach to Identify Lineage Guiding Molecules in Pericyte Cultures.

We report the use of self-assembled peptide (F2/S) hydrogels and cellular metabolomics to identify a number of innate molecules that are integral to the metabolic processes which drive cellular differentiation of multipotent pericyte stem cells. The culture system relies solely on substrate mechanics to induce differentiation in the absence of traditional differentiation media and therefore is a non-invasive approach to assessing cellular behavior at the molecular level and identifying key metabolites in this process. This novel approach demonstrates that simple metabolites can provide an alternative means to direct stem cell differentiation and that biomaterials can be used to identify them simply and quickly.

ATP induces contraction of cultured brain capillary pericytes via activation of P2Y-type purinergic receptors.

Brain capillary pericytes have been suggested to play a role in the regulation of cerebral blood flow under physiological and pathophysiological conditions. ATP has been shown to cause constriction of capillaries under ischemic conditions and suggested to be involved in the "no-reflow" phenomenon. To investigate the effects of extracellular ATP on pericyte cell contraction, we studied purinergic receptor activation of cultured bovine brain capillary pericytes. We measured intracellular Ca2+ concentration ([Ca2+]i) responses to purinergic agonists with the fluorescent indicators fura-2 and Cal-520 and estimated contraction of pericytes as relative change in cell area, using real-time confocal imaging. Addition of ATP caused an increase in cytosolic calcium and contraction of the brain capillary pericytes, both reversible and inhibited by the purinergic receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). Furthermore, we demonstrated that ATP-induced contraction could be eliminated by intracellular calcium chelation with BAPTA, indicating that the contraction was mediated via purinergic P2-type receptor-mediated [Ca2+]i signaling. ATP stimulation induced inositol triphosphate signaling, consistent with the notion of P2Y receptor activation. Receptor profiling studies demonstrated the presence of P2Y1 and P2Y2 receptors, using ATP, UTP, ADP, and the subtype specific agonists MRS2365 (P2Y1) and 2-thio-UTP (P2Y2). Addition of specific P2X agonists only caused an [Ca2+]i increase at high concentrations, attributed to activation of inositol triphosphate signaling. Our results suggest that contraction of brain capillary pericytes in vitro by activation of P2Y-type purinergic receptors is caused by intracellular calcium release. This adds more mechanistic understanding of the role of pericytes in vessel constriction and points toward P2Y receptors as potential therapeutic targets.NEW & NOTEWORTHY The study concerns brain capillary pericytes, which have been suggested to play a role in the regulation of cerebral blood flow. We show that extracellular ATP causes contraction of primary brain pericytes by stimulation of purinergic receptors and subsequent release of intracellular Ca2+ concentration ([Ca2+]i). The contraction is mainly mediated through activation of P2Y-receptor subtypes, including P2Y1 and P2Y2. These findings add more mechanistic understanding of the role of pericytes in regulation of capillary blood flow. ATP was earlier suggested to be involved in capillary constriction in brain pathologies, and our study gives a detailed account of a part of this important mechanism.

Capillary Networks for Bio-Artificial Three-Dimensional Tissues Fabricated Using Cell Sheet Based Tissue Engineering.

One of the most important challenges facing researchers in the field of regenerative medicine is to develop methods to introduce vascular networks into bioengineered tissues. Although cell scaffolds that slowly release angiogenic factors can promote post-transplantation angiogenesis, they cannot be used to construct thick tissues because of the time required for sufficient vascular network formation. Recently, the co-culture of graft tissue with vascular cells before transplantation has attracted attention as a way of promoting capillary angiogenesis. Although the co-cultured vascular cells can directly contribute to blood vessel formation within the tissue, a key objective that needs to be met is the construction of a continuous circulatory structure. Previously described strategies to reconstruct blood vessels include the culture of endothelial cells in a scaffold that contains microchannels or within the original vascular framework after decellularization of an entire organ. The technique, as developed by authors, involves the progressive stacking of three-layered cell sheets onto a vascular bed to induce the formation of a capillary network within the cell sheets. This approach enables the construction of thick, functional tissue of high cell density that can be transplanted by anastomosing its artery and vein (provided by the vascular bed) with host blood vessels.

WNT11-Conditioned Medium Promotes Angiogenesis through the Activation of Non-Canonical WNT-PKC-JNK Signaling Pathway.

BACKGROUND: We demonstrated that the transduction of Wnt11 into mesenchymal stem cells (MSCs) (MSCWnt11) promotes these cells differentiation into cardiac phenotypes. In the present study, we investigated the paracrine effects of MSCWnt11 on cardiac function and angiogenesis. METHODS AND RESULTS: Conditioned medium was collected from MSCWnt11 (CdMWnt11) and their control cells (CdMGFP). CdMWnt11, especially obtained from MSCWnt11 exposed to hypoxia, significantly promoted human umbilical vein endothelial cells (HUVECs) migration and increased capillary-like tube (CLT) formation, which was blocked by Wnt11 neutralizing antibody. Wnt11 protein was significantly higher in CdMWnt11 compared to that in CdMGFP. Directly treating HUVECs with recombinant Wnt11 protein significantly increased CLT formation, which was abrogated by treating cells with the JNK inhibitor SP600125, as well as the PKC inhibitor Calphostin-C. Moreover, the transfection of Wnt11 to HUVECs (HWnt11) significantly increased CLT formation and HUVEC migration, as well as upregulated p-pan-PKC and p-JNK expression. Injection of CdMWnt11 into the peri-infarct region in a rat acute myocardial infarction (AMI) model significantly improved cardiac function, reduced infarct size, and increased myocardial blood flow and blood vessel density in the ischemic area. CONCLUSION: Wnt11 released from MSCWnt11 increased angiogenesis and improved cardiac function via non-canonical Wnt-PKC-JNK dependent pathways.