Human atherosclerotic plaque transcriptomics reveals endothelial beta-2 spectrin as a potential regulator a leaky plaque microvasculature phenotype.

The presence of atherosclerotic plaque vessels is a critical factor in plaque destabilization. This may be attributable to the leaky phenotype of these microvessels, although direct proof for this notion is lacking. In this study, we investigated molecular and cellular patterns of stable and hemorrhaged human plaque to identify novel drivers of intraplaque vessel dysfunction. From transcriptome data of a human atherosclerotic lesion cohort, we reconstructed a co-expression network, identifying a gene module strongly and selectively correlated with both plaque microvascular density and inflammation. Spectrin Beta Non-Erythrocytic 1 (sptbn1) was identified as one of the central hubs of this module (along with zeb1 and dock1) and was selected for further study based on its predominant endothelial expression. Silencing of sptbn1 enhanced leukocyte transmigration and vascular permeability in vitro, characterized by an increased number of focal adhesions and reduced junctional VE-cadherin. In vivo, sptbn1 knockdown in zebrafish impaired the development of the caudal vein plexus. Mechanistically, increased substrate stiffness was associated with sptbn1 downregulation in endothelial cells in vitro and in human vessels. Plaque SPTBN1 mRNA and protein expression were found to correlate with an enhanced presence of intraplaque hemorrhage and future cardiovascular disease (CVD) events during follow-up. In conclusion, we identify SPTBN1 as a central hub gene in a gene program correlating with plaque vascularisation. SPTBN1 was regulated by substrate stiffness in vitro while silencing blocked vascular development in vivo, and compromised barrier function in vitro. Together, SPTBN1 is identified as a new potential regulator of the leaky phenotype of atherosclerotic plaque microvessels.

3D Niche-Inspired Scaffolds as a Stem Cell Delivery System for the Regeneration of the Osteochondral Interface.

The regeneration of the osteochondral unit represents a challenge due to the distinct cartilage and bone phases. Current strategies focus on the development of multiphasic scaffolds that recapitulate features of this complex unit and promote the differentiation of implanted bone-marrow derived stem cells (BMSCs). In doing so, challenges remain from the loss of stemness during in vitro expansion of the cells and the low control over stem cell activity at the interface with scaffolds in vitro and in vivo. Here, this work scaffolds inspired by the bone marrow niche that can recapitulate the natural healing process after injury. The construct comprises an internal depot of quiescent BMSCs, mimicking the bone marrow cavity, and an electrospun (ESP) capsule that "activates" the cells to migrate into an outer "differentiation-inducing" 3D printed unit functionalized with TGF-ß and BMP-2 peptides. In vitro, niche-inspired scaffolds retained a depot of nonproliferative cells capable of migrating and proliferating through the ESP capsule. Invasion of the 3D printed cavity results in location-specific cell differentiation, mineralization, secretion of alkaline phosphatase (ALP) and glycosaminoglycans (GAGs), and genetic upregulation of collagen II and collagen I. In vivo, niche-inspired scaffolds are biocompatible, promoted tissue formation in rat subcutaneous models, and regeneration of the osteochondral unit in rabbit models.

Capillary rarefaction: a missing link in renal and cardiovascular disease?

Patients with chronic kidney disease (CKD) have an increased risk for cardiovascular morbidity and mortality. Capillary rarefaction may be both one of the causes as well as a consequence of CKD and cardiovascular disease. We reviewed the published literature on human biopsy studies and conclude that renal capillary rarefaction occurs independently of the cause of renal function decline. Moreover, glomerular hypertrophy may be an early sign of generalized endothelial dysfunction, while peritubular capillary loss occurs in advanced renal disease. Recent studies with non-invasive measurements show that capillary rarefaction is detected systemically (e.g., in the skin) in individuals with albuminuria, as sign of early CKD and/or generalized endothelial dysfunction. Decreased capillary density is found in omental fat, muscle and heart biopsies of patients with advanced CKD as well as in skin, fat, muscle, brain and heart biopsies of individuals with cardiovascular risk factors. No biopsy studies have yet been performed on capillary rarefaction in individuals with early CKD. At present it is unknown whether individuals with CKD and cardiovascular disease merely share the same risk factors for capillary rarefaction, or whether there is a causal relationship between rarefaction in renal and systemic capillaries. Further studies on renal and systemic capillary rarefaction, including their temporal relationship and underlying mechanisms are needed. This review stresses the importance of preserving and maintaining capillary integrity and homeostasis in the prevention and management of renal and cardiovascular disease.

Identification of Compounds Protecting Pancreatic Islets against Oxidative Stress using a 3D Pseudoislet Screening Platform.

Oxidative stress leads to a lower success rate of clinical islet transplantation. Here, FDA-approved compounds are screened for their potential to decrease oxidative stress and to protect or enhance pancreatic islet viability and function. Studies are performed on in vitro "pseudoislet" spheroids, which are pre-incubated with 1280 different compounds and subjected to oxidative stress. Cell viability and oxidative stress levels are determined using a high-throughput fluorescence microscopy pipeline. Initial screening on cell viability results in 59 candidates. The top ten candidates are subsequently screened for their potential to decrease induced oxidative stress, and eight compounds efficient reduction of induced oxidative stress in both alpha and beta cells by 25-50%. After further characterization, the compound sulfisoxazole is found to be the most capable of reducing oxidative stress, also at short pre-incubation times, which is validated in primary human islets, where low oxidative stress levels and islet function are maintained. This study shows an effective screening strategy with 3D cell aggregates based on cell viability and oxidative stress, which leads to the discovery of several compounds with antioxidant capacity. The top candidate, sulfisoxazole is effective after a 30 min pre-incubation, maintains baseline islet function, and may help alleviate oxidative stress in pancreatic islets.

Endothelial ADAM10 controls cellular response to oxLDL and its deficiency exacerbates atherosclerosis with intraplaque hemorrhage and neovascularization in mice.

Introduction: The transmembrane protease A Disintegrin And Metalloproteinase 10 (ADAM10) displays a "pattern regulatory function," by cleaving a range of membrane-bound proteins. In endothelium, it regulates barrier function, leukocyte recruitment and angiogenesis. Previously, we showed that ADAM10 is expressed in human atherosclerotic plaques and associated with neovascularization. In this study, we aimed to determine the causal relevance of endothelial ADAM10 in murine atherosclerosis development in vivo. Methods and results: Endothelial Adam10 deficiency (Adam10 ecko ) in Western-type diet (WTD) fed mice rendered atherogenic by adeno-associated virus-mediated PCSK9 overexpression showed markedly increased atherosclerotic lesion formation. Additionally, Adam10 deficiency was associated with an increased necrotic core and concomitant reduction in plaque macrophage content. Strikingly, while intraplaque hemorrhage and neovascularization are rarely observed in aortic roots of atherosclerotic mice after 12 weeks of WTD feeding, a majority of plaques in both brachiocephalic artery and aortic root of Adam10ecko mice contained these features, suggestive of major plaque destabilization. In vitro, ADAM10 knockdown in human coronary artery endothelial cells (HCAECs) blunted the shedding of lectin-like oxidized LDL (oxLDL) receptor-1 (LOX-1) and increased endothelial inflammatory responses to oxLDL as witnessed by upregulated ICAM-1, VCAM-1, CCL5, and CXCL1 expression (which was diminished when LOX-1 was silenced) as well as activation of pro-inflammatory signaling pathways. LOX-1 shedding appeared also reduced in vivo, as soluble LOX-1 levels in plasma of Adam10ecko mice was significantly reduced compared to wildtypes. Discussion: Collectively, these results demonstrate that endothelial ADAM10 is atheroprotective, most likely by limiting oxLDL-induced inflammation besides its known role in pathological neovascularization. Our findings create novel opportunities to develop therapeutics targeting atherosclerotic plaque progression and stability, but at the same time warrant caution when considering to use ADAM10 inhibitors for therapy in other diseases.

Enhanced Microvasculature Formation and Patterning in iPSC-Derived Kidney Organoids Cultured in Physiological Hypoxia.

Stem cell-derived kidney organoids have been shown to self-organize from induced pluripotent stem cells into most important renal structures. However, the structures remain immature in culture and contain endothelial networks with low connectivity and limited organoid invasion. Furthermore, the nephrons lose their phenotype after approximately 25 days. To become applicable for future transplantation, further maturation in vitro is essential. Since kidneys in vivo develop in hypoxia, we studied the modulation of oxygen availability in culture. We hypothesized that introducing long-term culture at physiological hypoxia, rather than the normally applied non-physiological, hyperoxic 21% O2, could initiate angiogenesis, lead to enhanced growth factor expression and improve the endothelial patterning. We therefore cultured the kidney organoids at 7% O2 instead of 21% O2 for up to 25 days and evaluated nephrogenesis, growth factor expression such as VEGF-A and vascularization. Whole mount imaging revealed a homogenous morphology of the endothelial network with enhanced sprouting and interconnectivity when the kidney organoids were cultured in hypoxia. Three-dimensional vessel quantification confirmed that the hypoxic culture led to an increased average vessel length, likely due to the observed upregulation of VEGFA-189 and VEGFA-121, and downregulation of the antiangiogenic protein VEGF-A165b measured in hypoxia. This research indicates the importance of optimization of oxygen availability in organoid systems and the potential of hypoxic culture conditions in improving the vascularization of organoids.

Mesoporous Silica-Coated Gold Nanoparticles for Multimodal Imaging and Reactive Oxygen Species Sensing of Stem Cells.

Stem cell (SC)-based therapies hold the potential to revolutionize therapeutics by enhancing the body's natural repair processes. Currently, there are only three SC therapies with marketing authorization within the European Union. To optimize outcomes, it is important to understand the biodistribution and behavior of transplanted SCs in vivo. A variety of imaging agents have been developed to trace SCs; however, they mostly lack the ability to simultaneously monitor the SC function and biodistribution at high resolutions. Here, we report the synthesis and application of a nanoparticle (NP) construct consisting of a gold NP core coated with rhodamine B isothiocyanate (RITC)-doped mesoporous silica (AuMS). The MS layer further contained a thiol-modified internal surface and an amine-modified external surface for dye conjugation. Highly fluorescent AuMS of three different sizes were successfully synthesized. The NPs were non-toxic and efficiently taken up by limbal epithelial SCs (LESCs). We further showed that we can functionalize AuMS with a reactive oxygen species (ROS)-sensitive fluorescent dye using two methods, loading the probe into the mesopores, with or without additional capping by a lipid bilayer, and by covalent attachment to surface and/or mesoporous-functionalized thiol groups. All four formulations displayed a ROS concentration-dependent increase in fluorescence. Further, in an ex vivo SC transplantation model, a combination of optical coherence tomography and fluorescence microscopy was used to synergistically identify AuMS-labeled LESC distribution at micrometer resolution. Our AuMS constructs allow for multimodal imaging and simultaneous ROS sensing of SCs and represent a promising tool for in vivo SC tracing.

The response of three-dimensional pancreatic alpha and beta cell co-cultures to oxidative stress.

The pancreatic islets of Langerhans have low endogenous antioxidant levels and are thus especially sensitive to oxidative stress, which is known to influence cell survival and behaviour. As bioengineered islets are gaining interest for therapeutic purposes, it is important to understand how their composition can be optimized to diminish oxidative stress. We investigated how the ratio of the two main islet cell types (alpha and beta cells) and their culture in three-dimensional aggregates could protect against oxidative stress. Monolayer and aggregate cultures were established by seeding the alphaTC1 (alpha) and INS1E (beta) cell lines in varying ratios, and hydrogen peroxide was applied to induce oxidative stress. Viability, oxidative stress, and the level of the antioxidant glutathione were measured. Both aggregation and an increasing prevalence of INS1E cells in the co-cultures conferred greater resistance to cell death induced by oxidative stress. Increasing the prevalence of INS1E cells also decreased the number of alphaTC1 cells experiencing oxidative stress in the monolayer culture. In 3D aggregates, culturing the alphaTC1 and INS1E cells in a ratio of 50:50 prevented oxidative stress in both cell types. Together, the results of this study lead to new insight into how modulating the composition and dimensionality of a co-culture can influence the oxidative stress levels experienced by the cells.

Methodological approaches in aggregate formation and microscopic analysis to assess pseudoislet morphology and cellular interactions.

Microscopy has revolutionised our view on biology and has been vital for many discoveries since its invention around 200 years ago. Recent developments in cell biology have led to a strong interest in generating spheroids and organoids that better represent tissue. However, the current challenge faced by many researchers is the culture and analysis of these three-dimensional (3D) cell cultures. With the technological improvements in reconstructing volumetric datasets by optical sections, it is possible to quantify cells, their spatial arrangement, and the protein distribution without destroying the physical organization. We assessed three different microwell culture plates and four analysis tools for 3D imaging data for their applicability for the analysis of 3D cultures. A key advantage of microwell plates is their potential to perform high-throughput experiments in which cell cultures are generated and analysed in one single system. However, it was shown that this potential could be impacted by the material composition and microwell structure. For example, antibody staining was not possible in a hydrogel microwell, and truncated pyramid-structured microwells had increased background fluorescence due to their structure. Regarding analysis tools, four different software, namely CellProfiler, Fiji/ImageJ, Nikon GA3 and Imaris, were compared for their accuracy and applicability in analysing datasets from 3D cultures. The results showed that the open-access software, CellProfiler and Fiji, could quantify nuclei and cells, yet with varying results compared to manual counting, and may require post-processing optimisation. On the other hand, the GA3 and Imaris software packages showed excellent versatility in usage and accuracy in the quantification of nuclei and cells, and could classify cell localisation. Together these results provide critical considerations for microscopic imaging and analysis of 3D cell cultures.

Early bone ingrowth and segmental stability of a trussed titanium cage versus a polyether ether ketone cage in an ovine lumbar interbody fusion model.

BACKGROUND CONTEXT: Lumbar interbody fusion is an effective treatment for unstable spinal segments. However, the time needed to establish a solid bony interbody fusion between the two vertebrae may be longer than twelve months after surgery. During this time window, the instrumented spinal segment is assumed to be at increased risk for instability related complications such as cage migration or subsidence. It is hypothesized that the design of new interbody cages that enable direct osseointegration of the cage at the vertebral endplates, without requiring full bony fusion between the two vertebral endplates, might shorten the time window that the instrumented spinal segment is susceptible to failure. PURPOSE: To quantify the bone ingrowth and resulting segmental stability during consolidation of lumbar interbody fusion using two different cage types. STUDY DESIGN: Preclinical ovine model. METHODS: Seven skeletally mature sheep underwent bi-segmental lumbar interbody fusion surgery with one conventional polyether ether ketone (PEEK) cage, and one newly developed trussed titanium (TT) cage. After a postoperative time period of 13 weeks, non-destructive range of motion testing, and histologic analysis was performed. Additionally, sample specific finite element (FE) analysis was performed to predict the stability of the interbody fusion region alone. RESULTS: Physiological movement of complete spinal motion segments did not reveal significant differences between the segments operated with PEEK and TT cages. The onset of creeping substitution within the cage seemed to be sooner for PEEK cages, which led to significantly higher bone volume over total volume (BV/TV) compared with the TT cages. TT cages showed significantly more direct bone to implant contact (BIC). Although the mean stability of the interbody fusion region alone was not statistically different between the PEEK and TT cages, the variation within the cage types illustrated an all-or-nothing response for the PEEK cages while a more gradual increase in stability was found for the TT cages. CONCLUSIONS: Spinal segments operated with conventional PEEK cages were not different from those operated with newly developed TT cages in terms of segmental stability but did show a different mechanism of bone ingrowth and attachment. Based on the differences in development of bony fusion, we hypothesize that TT cages might facilitate increased early segmental stability by direct osseointegration of the cage at the vertebral endplates without requiring complete bony bridging through the cage. CLINICAL SIGNIFICANCE: Interbody cage type affects the consolidation process of spinal interbody fusion. Whether different consolidation processes of spinal interbody fusion result in clinically significant differences requires further investigation.

Thiol-ene cross-linked alginate hydrogel encapsulation modulates the extracellular matrix of kidney organoids by reducing abnormal type 1a1 collagen deposition.

Differentiated kidney organoids from induced pluripotent stem cells hold promise as a treatment for patients with kidney diseases. Before these organoids can be translated to the clinic, shortcomings regarding their cellular and extracellular compositions, and their developmental plateau need to be overcome. We performed a proteomic analysis on kidney organoids cultured for a prolonged culture time and we found a specific change in the extracellular matrix composition with increased expression of types 1a1, 2 and 6a1 collagen. Such an excessive accumulation of specific collagen types is a hallmark of renal fibrosis that causes a life-threatening pathological condition by compromising key functions of the human kidney. Here we hypothesized the need for a three-dimensional environment to grow the kidney organoids, which could better mimic the in vivo surroundings of the developing kidney than standard culture on an air-liquid interface. Encapsulating organoids for four days in a soft, thiol-ene cross-linked alginate hydrogel resulted in decreased type 1a1 collagen expression. Furthermore, the encapsulation did not result in any changes of organoid structural morphology. Using a biomaterial to modulate collagen expression allows for a prolonged kidney organoid culture in vitro and a reduction of abnormal type 1a1 collagen expression bringing kidney organoids closer to clinical application.

Mimicking the Human Tympanic Membrane: The Significance of Scaffold Geometry.

The human tympanic membrane (TM) captures sound waves from the environment and transforms them into mechanical motion. The successful transmission of these acoustic vibrations is attributed to the unique architecture of the TM. However, a limited knowledge is available on the contribution of its discrete anatomical features, which is important for fabricating functional TM replacements. This work synergizes theoretical and experimental approaches toward understanding the significance of geometry in tissue-engineered TM scaffolds. Three test designs along with a plain control are chosen to decouple some of the dominant structural elements, such as the radial and circumferential alignment of the collagen fibrils. In silico models suggest a geometrical dependency of their mechanical and acoustical responses, where the presence of radially aligned fibers is observed to have a more prominent effect compared to their circumferential counterparts. Following which, a hybrid fabrication strategy combining electrospinning and additive manufacturing has been optimized to manufacture biomimetic scaffolds within the dimensions of the native TM. The experimental characterizations conducted using macroindentation and laser Doppler vibrometry corroborate the computational findings. Finally, biological studies with human dermal fibroblasts and human mesenchymal stromal cells reveal a favorable influence of scaffold hierarchy on cellular alignment and subsequent collagen deposition.

ADAM10-Mediated Cleavage of ICAM-1 Is Involved in Neutrophil Transendothelial Migration.

To efficiently cross the endothelial barrier during inflammation, neutrophils first firmly adhere to the endothelial surface using the endothelial adhesion molecule ICAM-1. Upon actual transmigration, the release from ICAM-1 is required. While Integrin LFA1/Mac1 de-activation is one described mechanism that leads to this, direct cleavage of ICAM-1 from the endothelium represents a second option. We found that a disintegrin and metalloprotease 10 (ADAM10) cleaves the extracellular domain of ICAM-1 from the endothelial surface. Silencing or inhibiting endothelial ADAM10 impaired the efficiency of neutrophils to cross the endothelium, suggesting that neutrophils use endothelial ADAM10 to dissociate from ICAM-1. Indeed, when measuring transmigration kinetics, neutrophils took almost twice as much time to finish the diapedesis step when ADAM10 was silenced. Importantly, we found increased levels of ICAM-1 on the transmigrating neutrophils when crossing an endothelial monolayer where such increased levels were not detected when neutrophils crossed bare filters. Using ICAM-1-GFP-expressing endothelial cells, we show that ICAM-1 presence on the neutrophils can also occur by membrane transfer from the endothelium to the neutrophil. Based on these findings, we conclude that endothelial ADAM10 contributes in part to neutrophil transendothelial migration by cleaving ICAM-1, thereby supporting the release of neutrophils from the endothelium during the final diapedesis step.

Bone Marrow Harbors a Unique Population of Dendritic Cells with the Potential to Boost Neutrophil Formation upon Exposure to Fungal Antigen.

Apart from controlling hematopoiesis, the bone marrow (BM) also serves as a secondary lymphoid organ, as it can induce naïve T cell priming by resident dendritic cells (DC). When analyzing DCs in murine BM, we uncovered that they are localized around sinusoids, can (cross)-present antigens, become activated upon intravenous LPS-injection, and for the most part belong to the cDC2 subtype which is associated with Th2/Th17 immunity. Gene-expression profiling revealed that BM-resident DCs are enriched for several c-type lectins, including Dectin-1, which can bind beta-glucans expressed on fungi and yeast. Indeed, DCs in BM were much more efficient in phagocytosis of both yeast-derived zymosan-particles and Aspergillus conidiae than their splenic counterparts, which was highly dependent on Dectin-1. DCs in human BM could also phagocytose zymosan, which was dependent on ß1-integrins. Moreover, zymosan-stimulated BM-resident DCs enhanced the differentiation of hematopoietic stem and progenitor cells towards neutrophils, while also boosting the maintenance of these progenitors. Our findings signify an important role for BM DCs as translators between infection and hematopoiesis, particularly in anti-fungal immunity. The ability of BM-resident DCs to boost neutrophil formation is relevant from a clinical perspective and contributes to our understanding of the increased susceptibility for fungal infections following BM damage.

Hematopoietic stem and progenitor cells use podosomes to transcellularly cross the bone marrow endothelium.

Bone marrow endothelium plays an important role in the homing of hematopoietic stem and progenitor cells upon transplantation, but surprisingly little is known on how the bone marrow endothelial cells regulate local permeability and hematopoietic stem and progenitor cells transmigration. We show that temporal loss of vascular endothelial-cadherin function promotes vascular permeability in BM, even upon low-dose irradiation. Loss of vascular endothelial-cadherin function also enhances homing of transplanted hematopoietic stem and progenitor cells to the bone marrow of irradiated mice although engraftment is not increased. Intriguingly, stabilizing junctional vascular endothelial-cadherin in vivo reduced bone marrow permeability, but did not prevent hematopoietic stem and progenitor cells migration into the bone marrow, suggesting that hematopoietic stem and progenitor cells use the transcellular migration route to enter the bone marrow. Indeed, using an in vitro migration assay, we show that human hematopoietic stem and progenitor cells predominantly cross bone marrow endothelium in a transcellular manner in homeostasis by inducing podosome-like structures. Taken together, vascular endothelial-cadherin is crucial for BM vascular homeostasis but dispensable for the homing of hematopoietic stem and progenitor cells. These findings are important in the development of potential therapeutic targets to improve hematopoietic stem and progenitor cell homing strategies.

Transport and Preservation Comparison of Preloaded and Prestripped-Only DMEK Grafts.

PURPOSE: This study compares the effect of the transport of conventionally prestripped Descemet membrane endothelial keratoplasty (DMEK) tissue with the DMEK revolutionary advanced Preloadable Injection Device (RAPID) preloaded transport system from Geuder AG (Heidelberg, Germany). Endothelial cell loss, tissue integrity, endothelial cell phenotype, and viability were assessed and compared. METHODS: Twelve DMEK grafts were prestripped by the cornea bank and transported using the following 2 conditions: conventional flask (n = 6) or a preloaded transport cartridge (DMEK RAPID, n = 6). After transport, tissues were analyzed for cell density; denuded areas; immunolocalization of corneal endothelial markers, such as ZO-1, CD166, and Na/K ATPase; histology analysis; and cell viability staining with Hoechst, calcein AM, and ethidium homodimer. RESULTS: Endothelial cell loss (10.35% vs. 9.15%) did not differ between transport conditions. Histological analysis confirmed the integrity of the Descemet membrane and endothelial cell layer with both transport conditions. Similarly, the corneal endothelial cell mosaic was conserved in both conditions. The ZO-1 tight junctions confirmed the integrity of the confluent corneal endothelial cell monolayer. CD166 and Na/K ATPase detection with immunofluorescence was also comparable. A similar percentage of dead cells was reported in both conditions (18.1% vs. 16.73%). Moreover, the surface covered with calcein-positive cells (59.02% vs. 61.95%) did not differ between transport conditions. CONCLUSIONS: Our results suggest that DMEK grafts can be prestripped or preloaded into a novel transport cartridge and shipped to the clinic with comparable endothelial cell loss, phenotypical marker expression, and viability to the conventional prestripped donor tissue.

LIM-only protein FHL2 attenuates vascular tissue factor activity, inhibits thrombus formation in mice and FHL2 genetic variation associates with human venous thrombosis.

Bleeding disorders and thrombotic complications are major causes of morbidity and mortality with many cases being unexplained. Thrombus formation involves aberrant expression and activation of tissue factor (TF) in vascular endothelial and smooth muscle cells. Here, we sought to identify factors that modulate TF gene expression and activity in these vascular cells. The LIM-only protein FHL2 is a scaffolding protein that modulates signal transduction pathways with crucial functions in endothelial and smooth muscle cells. However, the role of FHL2 in TF regulation and thrombosis remains unexplored. Using a murine model of venous thrombosis in mesenteric vessels, we demonstrated that FHL2 deficiency results in exacerbated thrombus formation. Gain- and loss-of-function experiments revealed that FHL2 represses TF expression in endothelial and smooth muscle cells through inhibition of the transcription factors nuclear factor κB and activating protein-1. Furthermore, we observed that FHL2 interacts with the cytoplasmic tail of TF. In line with our in vivo observations, FHL2 decreases TF activity in endothelial and smooth muscle cells whereas FHL2 knockdown or deficiency results in enhanced TF activity. Finally, the FHL2 single nucleotide polymorphism rs4851770 was associated with the risk of venous thrombosis in a large population of venous thrombosis cases and control subjects from 12 studies (INVENT consortium). Altogether, our results highlight functional involvement of FHL2 in TF-mediated coagulation and identify FHL2 as a novel gene associated with venous thrombosis in humans.

Oxygen and nutrient delivery in tissue engineering: Approaches to graft vascularization.

The field of tissue engineering is making great strides in developing replacement tissue grafts for clinical use, marked by the rapid development of novel biomaterials, their improved integration with cells, better-directed growth and differentiation of cells, and improved three-dimensional tissue mass culturing. One major obstacle that remains, however, is the lack of graft vascularization, which in turn renders many grafts to fail upon clinical application. With that, graft vascularization has turned into one of the holy grails of tissue engineering, and for the majority of tissues, it will be imperative to achieve adequate vascularization if tissue graft implantation is to succeed. Many different approaches have been developed to induce or augment graft vascularization, both in vitro and in vivo. In this review, we highlight the importance of vascularization in tissue engineering and outline various approaches inspired by both biology and engineering to achieve and augment graft vascularization.

CXCR4, but not CXCR3, drives CD8+ T-cell entry into and migration through the murine bone marrow.

The BM serves as a blood-forming organ, but also supports the maintenance and immune surveillance function of many T cells. Yet, in contrast to other organs, little is known about the molecular mechanisms that drive T-cell migration to and localization inside the BM. As BM accumulates many CXCR3-expressing memory CD8+ T cells, we tested the involvement of this chemokine receptor, but found that CXCR3 is not required for BM entry. In contrast, we could demonstrate that CXCR4, which is highly expressed on both naive and memory CD8+ T cells in BM, is critically important for homing of all CD8+ T-cell subsets to the BM in mice. Upon entry into the BM parenchyma, both naïve and memory CD8+ T cells locate close to sinusoidal vessels. Intravital imaging experiments revealed that CD8 T cells are surprisingly immobile and we found that they interact with ICAM-1+VCAM-1+BP-1+ perivascular stromal cells. These cells are the major source of CXCL12, but also express key survival factors and maintenance cytokines IL-7 and IL-15. We therefore conclude that CXCR4 is not only crucial for entry of CD8+ T cells into the BM, but also controls their subsequent localization toward BM niches that support their survival.

Multi-photon microscopy in cardiovascular research.

Multiphoton laser scanning microscopy has proven profound value for ex vivo 3D histology and in vivo imaging of motionless tissue. The development of triggering systems and fast imaging methods, combined with advanced preparation procedures solved the challenging task of intravital imaging of the fast pulsating heart and major arteries in animals and further increased the popularity of intravital multiphoton imaging in cardiovascular research. This review article will highlight the potential of multiphoton microscopy for the visualization and characterization of dynamical and structural processes involved in cardiac and vascular diseases, both in an ex vivo and an intravital animal setting. Examples will be given how multiphoton microscopy can be applied to imaging of atherosclerotic plaque development and progression at subcellular level as well as to intravital imaging of inflammatory processes in the heart. In addition to highlighting the potential of multiphoton microscopy in preclinical cardiovascular research, we will discuss how this tool and its applications may be clinically translated to support disease diagnosis and therapy in patients.