Lung lymphatic endothelial cells undergo inflammatory and prothrombotic changes in a model of chronic obstructive pulmonary disease.

The lymphatic vasculature regulates lung homeostasis through drainage of fluid and trafficking of immune cells and plays a key role in the response to lung injury in several disease states. We have previously shown that lymphatic dysfunction occurs early in the pathogenesis of chronic obstructive pulmonary disease (COPD) caused by cigarette smoke (CS) and that this is associated with increased thrombin and fibrin clots in lung lymph. However, the direct effects of CS and thrombin on lymphatic endothelial cells (LECs) in COPD are not entirely clear. Studies of the blood vasculature have shown that COPD is associated with increased thrombin after CS exposure that causes endothelial dysfunction characterized by changes in the expression of coagulation factors and leukocyte adhesion proteins. Here, we determined whether similar changes occur in LECs. We used an in vitro cell culture system and treated human lung microvascular lymphatic endothelial cells with cigarette smoke extract (CSE) and/or thrombin. We found that CSE treatment led to decreased fibrinolytic activity in LECs, which was associated with increased expression of plasminogen activator inhibitor 1 (PAI-1). LECs treated with both CSE and thrombin together had a decreased expression of tissue factor pathway inhibitor (TFPI) and increased expression of adhesion molecules. RNA sequencing of lung LECs isolated from mice exposed to CS also showed upregulation of prothrombotic and inflammatory pathways at both acute and chronic exposure time points. Analysis of publicly available single-cell RNA sequencing of LECs as well as immunohistochemical staining of lung tissue from COPD patients supported these data and showed increased expression of inflammatory markers in LECs from COPD patients compared to those from controls. These studies suggest that in parallel with blood vessels, the lymphatic endothelium undergoes inflammatory changes associated with CS exposure and increased thrombin in COPD. Further research is needed to unravel the mechanisms by which these changes affect lymphatic function and drive tissue injury in COPD.

Lymphatic Dysfunction Models an Autoimmune Emphysema Phenotype of Chronic Obstructive Pulmonary Disease.

Chronic Obstructive Pulmonary Disease (COPD) is a heterogeneous disease that is characterized by many clinical phenotypes. One such phenotype of COPD is defined by emphysema, pathogenic lung tertiary lymphoid organs (TLOs), and autoantibody production. We have previously shown that lymphatic dysfunction can cause lung TLO formation and lung injury in mice. We now sought to uncover whether underlying lymphatic dysfunction may be a driver of lung injury in cigarette smoke (CS)-induced COPD. We found that lung TLOs in mice with lymphatic dysfunction produce autoantibodies and are associated with a lymphatic endothelial cell subtype that expresses antigen presentation genes. Mice with underlying lymphatic dysfunction develop increased emphysema after CS exposure, with increased size and activation of TLOs. CS further increased autoantibody production in mice with lymphatic dysfunction. B-cell blockade prevented TLO formation and decreased lung injury after CS in mice with lymphatic dysfunction. Using tissue from human COPD patients, we also found evidence of a lymphatic gene signature that was specific to patients with emphysema and prominent TLOs compared to COPD patients without emphysema. Taken together, these data suggest that lymphatic dysfunction may underlie lung injury in a subset of COPD patients with an autoimmune emphysema phenotype.

Specification of fetal liver endothelial progenitors to functional zonated adult sinusoids requires c-Maf induction.

The liver vascular network is patterned by sinusoidal and hepatocyte co-zonation. How intra-liver vessels acquire their hierarchical specialized functions is unknown. We study heterogeneity of hepatic vascular cells during mouse development through functional and single-cell RNA-sequencing. The acquisition of sinusoidal endothelial cell identity is initiated during early development and completed postnatally, originating from a pool of undifferentiated vascular progenitors at E12. The peri-natal induction of the transcription factor c-Maf is a critical switch for the sinusoidal identity determination. Endothelium-restricted deletion of c-Maf disrupts liver sinusoidal development, aberrantly expands postnatal liver hematopoiesis, promotes excessive postnatal sinusoidal proliferation, and aggravates liver pro-fibrotic sensitivity to chemical insult. Enforced c-Maf overexpression in generic human endothelial cells switches on a liver sinusoidal transcriptional program that maintains hepatocyte function. c-Maf represents an inducible intra-organotypic and niche-responsive molecular determinant of hepatic sinusoidal cell identity and lays the foundation for the strategies for vasculature-driven liver repair.

Cooperative ETS Transcription Factors Enforce Adult Endothelial Cell Fate and Cardiovascular Homeostasis.

Current dogma dictates that during adulthood, endothelial cells (ECs) are locked in an immutable stable homeostatic state. By contrast, herein we show that maintenance of EC fate and function are linked and active processes, which depend on the constitutive cooperativity of only two ETS-transcription factors (TFs) ERG and Fli1. While deletion of either Fli1 or ERG manifest subtle vascular dysfunction, their combined genetic deletion in adult EC results in acute vasculopathy and multiorgan failure, due to loss of EC fate and integrity, hyperinflammation, and spontaneous thrombosis, leading to death. ERG and Fli1 co-deficiency cause rapid transcriptional silencing of pan- and organotypic vascular core genes, with dysregulation of inflammation and coagulation pathways. Vascular hyperinflammation leads to impaired hematopoiesis with myeloid skewing. Accordingly, enforced ERG and FLI1 expression in adult human mesenchymal stromal cells activates vascular programs and functionality enabling engraftment of perfusable vascular network. GWAS-analysis identified vascular diseases are associated with FLI1/Erg mutations. Constitutive expression of ERG and Fli1 uphold EC fate, physiological function, and resilience in adult vasculature; while their functional loss can contribute to systemic human diseases.

Human Induced Pluripotent Stem Cell-Derived Brain Endothelial Cells: Current Controversies.

Brain microvascular endothelial cells (BMECs) possess unique properties that are crucial for many functions of the blood-brain-barrier (BBB) including maintenance of brain homeostasis and regulation of interactions between the brain and immune system. The generation of a pure population of putative brain microvascular endothelial cells from human pluripotent stem cell sources (iBMECs) has been described to meet the need for reliable and reproducible brain endothelial cells in vitro. Human pluripotent stem cells (hPSCs), embryonic or induced, can be differentiated into large quantities of specialized cells in order to study development and model disease. These hPSC-derived iBMECs display endothelial-like properties, such as tube formation and low-density lipoprotein uptake, high transendothelial electrical resistance (TEER), and barrier-like efflux transporter activities. Over time, the de novo generation of an organotypic endothelial cell from hPSCs has aroused controversies. This perspective article highlights the developments made in the field of hPSC derived brain endothelial cells as well as where experimental data are lacking, and what concerns have emerged since their initial description.

Pluripotent stem cell-derived epithelium misidentified as brain microvascular endothelium requires ETS factors to acquire vascular fate.

Cells derived from pluripotent sources in vitro must resemble those found in vivo as closely as possible at both transcriptional and functional levels in order to be a useful tool for studying diseases and developing therapeutics. Recently, differentiation of human pluripotent stem cells (hPSCs) into brain microvascular endothelial cells (ECs) with blood-brain barrier (BBB)-like properties has been reported. These cells have since been used as a robust in vitro BBB model for drug delivery and mechanistic understanding of neurological diseases. However, the precise cellular identity of these induced brain microvascular endothelial cells (iBMECs) has not been well described. Employing a comprehensive transcriptomic metaanalysis of previously published hPSC-derived cells validated by physiological assays, we demonstrate that iBMECs lack functional attributes of ECs since they are deficient in vascular lineage genes while expressing clusters of genes related to the neuroectodermal epithelial lineage (Epi-iBMEC). Overexpression of key endothelial ETS transcription factors (ETV2, ERG, and FLI1) reprograms Epi-iBMECs into authentic endothelial cells that are congruent with bona fide endothelium at both transcriptomic as well as some functional levels. This approach could eventually be used to develop a robust human BBB model in vitro that resembles the human brain EC in vivo for functional studies and drug discovery.

Personalized Fiber-Reinforcement Networks for Meniscus Reconstruction.

The menisci are fibrocartilaginous tissues that are crucial to the load-sharing and stability of the knee, and when injured, these properties are compromised. Meniscus replacement scaffolds have utilized the circumferential alignment of fibers to recapitulate the microstructure of the native meniscus; however, specific consideration of size, shape, and morphology has been largely overlooked. The purpose of this study was to personalize the fiber-reinforcement network of a meniscus reconstruction scaffold. Human cadaveric menisci were measured for a host of tissue (length, width) and subtissue (regional widths, root locations) properties, which all showed considerable variability between donors. Next, the asymmetrical fiber network was optimized to minimize the error between the dimensions of measured menisci and predicted fiber networks, providing a 51.0% decrease (p = 0.0091) in root-mean-square (RMS) error. Finally, a separate set of human cadaveric knees was obtained, and donor-specific fiber-reinforced scaffolds were fabricated. Under cyclic loading for load-distribution analysis, in situ implantation of personalized scaffolds following total meniscectomy restored contact area (253.0 mm2 to 488.9 mm2, p = 0.0060) and decreased contact stress (1.96 MPa to 1.03 MPa, p = 0.0025) to near-native values (597.4 mm2 and 0.83 MPa). Clinical use of personalized meniscus devices that restore physiologic contact stress distributions may prevent the development of post-traumatic osteoarthritis following meniscal injury.

Tumour exosomal CEMIP protein promotes cancer cell colonization in brain metastasis.

The development of effective therapies against brain metastasis is currently hindered by limitations in our understanding of the molecular mechanisms driving it. Here we define the contributions of tumour-secreted exosomes to brain metastatic colonization and demonstrate that pre-conditioning the brain microenvironment with exosomes from brain metastatic cells enhances cancer cell outgrowth. Proteomic analysis identified cell migration-inducing and hyaluronan-binding protein (CEMIP) as elevated in exosomes from brain metastatic but not lung or bone metastatic cells. CEMIP depletion in tumour cells impaired brain metastasis, disrupting invasion and tumour cell association with the brain vasculature, phenotypes rescued by pre-conditioning the brain microenvironment with CEMIP+ exosomes. Moreover, uptake of CEMIP+ exosomes by brain endothelial and microglial cells induced endothelial cell branching and inflammation in the perivascular niche by upregulating the pro-inflammatory cytokines encoded by Ptgs2, Tnf and Ccl/Cxcl, known to promote brain vascular remodelling and metastasis. CEMIP was elevated in tumour tissues and exosomes from patients with brain metastasis and predicted brain metastasis progression and patient survival. Collectively, our findings suggest that targeting exosomal CEMIP could constitute a future avenue for the prevention and treatment of brain metastasis.

Biomechanical characterization of a novel collagen-hyaluronan infused 3D-printed polymeric device for partial meniscus replacement.

The menisci transmit load by increasing the contact area and decreasing peak contact stresses on the articular surfaces. Meniscal lesions are among the most common orthopedic injuries, and resulting meniscectomies are associated with adverse polycaprolactone contact mechanics changes and, ultimately, an increased likelihood of osteoarthritis. Meniscus scaffolds were fabricated by 3D-printing a network of circumferential and radial filaments of resorbable polymer (poly(desaminotyrosyl-tyrosine dodecyl ester dodecanoate)) and infused with collagen-hyaluronan. The scaffold demonstrated an instantaneous compressive modulus (1.66 ± 0.44 MPa) comparable to native meniscus (1.52 ± 0.59 MPa). The scaffold aggregate modulus (1.33 ± 0.51 MPa) was within 2% of the native value (1.31 ± 0.36 MPa). In tension, the scaffold displayed a comparable stiffness to native tissue (127.6-97.1 N/mm) and an ultimate load of 33% of the native value. Suture pull-out load of scaffolds (83.1 ± 10.0 N) was within 10% of native values (91.5 ± 15.4 N). Contact stress analysis demonstrated the scaffold reduced peak contact stress by 60-67% and increased contact area by 38%, relative to partial meniscectomy. This is the first meniscal scaffold to match both the axial compressive properties and the circumferential tensile stiffness of the native meniscus. The improvement of joint contact mechanics, relative to partial meniscectomy alone, motivates further investigation using a large animal model. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B:2457-2465, 2019.

In vitro conversion of adult murine endothelial cells to hematopoietic stem cells.

The ability to generate hematopoietic stem cells (HSCs) in vitro would have an immeasurable impact on many areas of clinical practice, including trauma, cancer, and congenital disease. In this protocol, we describe a stepwise approach that converts adult murine endothelial cells (ECs) to HSCs, termed 'reprogrammed ECs into hematopoietic stem and progenitor cells' (rEC-HSPCs). The conversion, which is achieved without cells transitioning through a pluripotent state, comprises three phases: induction, specification, and expansion. Adult ECs are first isolated from Runx1-IRES-GFP; Rosa26-rtTa mice and maintained in culture under EC growth factor stimulation and Tgfß inhibition. In the first (induction) phase of conversion (days 0-8), four transcription factors (TFs)-FosB, Gfi1, Runx1, and Spi1 (FGRS)-are expressed transiently, which results in endogenous Runx1 expression. During the second (specification) phase (days 8-20), endogenous Runx1+ FGRS-transduced ECs commit to a hematopoietic fate and no longer require exogenous FGRS expression. Finally, the vascular niche drives robust proliferation of rEC-HSPCs during the expansion phase (days 20-28). The resulting converted cells possess a transcriptomic signature and long-term self-renewal capacity indistinguishable from those of adult HSCs. In this protocol, we also describe functional in vitro and in vivo assays that can be used to demonstrate that rEC-HSPCs are competent for clonal engraftment and possess multi-lineage reconstitution potential, including antigen-dependent adaptive immune function. This approach thus provides a tractable strategy for interrogating the generation of engraftable hematopoietic cells, advancing the mechanistic understanding of hematopoietic development and HSC self-renewal.