Cyclosporine Induces Fenestra-Associated Injury in Human Renal Microvessels In Vitro.

The use of cyclosporine A (CsA) in transplantation is frequently associated with nephrotoxicity, characterized by renal vascular injury, thrombotic microangiopathy, and striped interstitial fibrosis. Here, using human kidney-specific microvascular endothelial cells (HKMECs), we showed that CsA inhibited NFAT1 activation and impaired VEGF signaling in these ECs in a dose- and time-dependent manner. Integrated genome regulatory analyses identified key distinctions in the landscapes of HKMECs compared to human umbilical vein endothelial cells, particularly around genes related to the formation and maintenance of fenestrae. Using a bioengineered flow-directed 3D kidney microphysiological system, we revealed that CsA-induced kidney microvascular injury was associated with fenestrae and cell adhesion impairment, membrane swelling, and erythrocyte adhesion and extravasation into the interstitial space. Our data provide novel insights into kidney-specific molecular and structural mechanisms of CsA-induced microvascular injury. Our results also suggest VEGF-related pathways as potential targets for therapy during CsA treatment and emphasize the importance of leveraging species and organ-specific cells to better reflect human pathophysiology and the response to injury.

Genetic variation implicates plasma angiopoietin-2 in the development of acute kidney injury sub-phenotypes.

BACKGROUND: We previously identified two acute kidney injury (AKI) sub-phenotypes (AKI-SP1 and AKI-SP2) with different risk of poor clinical outcomes and response to vasopressor therapy. Plasma biomarkers of endothelial dysfunction (tumor necrosis factor receptor-1, angiopoietin-1 and 2) differentiated the AKI sub-phenotypes. However, it is unknown whether these biomarkers are simply markers or causal mediators in the development of AKI sub-phenotypes. METHODS: We tested for associations between single-nucleotide polymorphisms within the Angiopoietin-1, Angiopoietin-2, and Tumor Necrosis Factor Receptor 1A genes and AKI- SP2 in 421 critically ill subjects of European ancestry. Top performing single-nucleotide polymorphisms (FDR < 0.05) were tested for cis-biomarker expression and whether genetic risk for AKI-SP2 is mediated through circulating biomarkers. We also completed in vitro studies using human kidney microvascular endothelial cells. Finally, we calculated the renal clearance of plasma biomarkers using 20 different timed urine collections. RESULTS: A genetic variant, rs2920656C > T, near ANGPT2 was associated with reduced risk of AKI-SP2 (odds ratio, 0.45; 95% CI, 0.31-0.66; adjusted FDR = 0.003) and decreased plasma angiopoietin-2 (p = 0.002). Causal inference analysis showed that for each minor allele (T) the risk of developing AKI-SP2 decreases by 16%. Plasma angiopoietin-2 mediated 41.5% of the rs2920656 related risk for AKI-SP2. Human kidney microvascular endothelial cells carrying the T allele of rs2920656 produced numerically lower levels of angiopoietin-2 although this was not statistically significant (p = 0.07). Finally, analyses demonstrated that angiopoietin-2 is minimally renally cleared in critically ill subjects. CONCLUSION: Genetic mediation analysis provides supportive evidence that angiopoietin-2 plays a causal role in risk for AKI-SP2.

Open microfluidic coculture reveals paracrine signaling from human kidney epithelial cells promotes kidney specificity of endothelial cells.

Endothelial cells (ECs) from different human organs possess organ-specific characteristics that support specific tissue regeneration and organ development. EC specificity is identified by both intrinsic and extrinsic cues, among which the parenchyma and organ-specific microenvironment are critical contributors. These extrinsic cues are, however, largely lost during ex vivo cultures. Outstanding challenges remain to understand and reestablish EC organ specificity for in vitro studies to recapitulate human organ-specific physiology. Here, we designed an open microfluidic platform to study the role of human kidney tubular epithelial cells in supporting EC specificity. The platform consists of two independent cell culture regions segregated with a half wall; culture media are added to connect the two culture regions at a desired time point, and signaling molecules can travel across the half wall (paracrine signaling). Specifically, we report that in the microscale coculture device, primary human kidney proximal tubule epithelial cells (HPTECs) rescued primary human kidney peritubular microvascular EC (HKMEC) monolayer integrity and fenestra formation and that HPTECs upregulated key HKMEC kidney-specific genes (hepatocyte nuclear factor 1 homeobox B, adherens junctions-associated protein 1, and potassium voltage-gated channel subfamily J member 16) and endothelial activation genes (vascular cell adhesion molecule-1, matrix metalloproteinase-7, and matrix metalloproteinase-10) in coculture. Coculturing with HPTECs also promoted kidney-specific genotype expression in human umbilical vein ECs and human pluripotent stem cell-derived ECs. Compared with culture in HPTEC conditioned media, coculture of ECs with HPTECs showed increased upregulation of kidney-specific genes, suggesting potential bidirectional paracrine signaling. Importantly, our device is compatible with standard pipettes, incubators, and imaging readouts and could also be easily adapted to study cell signaling between other rare or sensitive cells.

Transforming Endothelium with Platelet-Rich Plasma in Engineered Microvessels.

Vascularization remains an obstacle when engineering complex tissues for regeneration and disease modeling. Although progress has been made in recreating 3D vascular structures, challenges exist in generating a mature, functional endothelium. It is demonstrated that perfusing engineered microvessels with platelet-rich plasma, a critical homeostatic component in vivo that is often overlooked in vitro, substantially transforms the endothelium, both maturing endothelial cells and improving functionality in 24 h. Platelets readily adhered to the exposed collagen-I substrate through small gaps within engineered vessels without forming thrombi. The adherent platelets improve barrier function, enhance endothelial glycolysis, reduce thrombogenicity, and enrich smooth muscle cell growth surrounding the endothelium. These findings demonstrate that platelets are essential to the function of endothelium during vascular maturation and remodeling. This study sheds light on a potential strategy to engineer stable, implantable vascular networks.

Binding Heterogeneity of Plasmodium falciparum to Engineered 3D Brain Microvessels Is Mediated by EPCR and ICAM-1.

Cerebral malaria is a severe neurological complication associated with sequestration of Plasmodium falciparum-infected erythrocytes (IE) in the brain microvasculature, but the specific binding interactions remain under debate. Here, we have generated an engineered three-dimensional (3D) human brain endothelial microvessel model and studied P. falciparum binding under the large range of physiological flow velocities that occur in both health and disease. Perfusion assays on 3D microvessels reveal previously unappreciated phenotypic heterogeneity in parasite binding to tumor necrosis factor alpha (TNF-α)-activated brain endothelial cells. While clonal parasite lines expressing a group B P. falciparum erythrocyte membrane protein 1 (PfEMP1) present an increase in binding to activated 3D microvessels, P. falciparum-IE expressing DC8-PfEMP1 present a decrease in binding. The differential response to endothelium activation is mediated by surface expression changes of endothelial protein C receptor (EPCR) and intercellular adhesion molecule 1 (ICAM-1). These findings demonstrate heterogeneity in parasite binding and provide evidence for a parasite strategy to adapt to a changing microvascular environment during infection. The engineered 3D human brain microvessel model provides new mechanistic insight into parasite binding and opens opportunities for further studies on malaria pathogenesis and parasite-vessel interactions.IMPORTANCE Cerebral malaria research has been hindered by the inaccessibility of the brain. Here, we have developed an engineered 3D human brain microvessel model that mimics the blood flow rates and architecture of small blood vessels to study how P. falciparum-infected human erythrocytes attach to brain endothelial cells. By studying parasite lines with different adhesive properties, we show that the malaria parasite binding rate is heterogeneous and strongly influenced by physiological differences in flow and whether the endothelium has been previously activated by TNF-α, a proinflammatory cytokine that is linked to malaria disease severity. We also show the importance of human EPCR and ICAM-1 in parasite binding. Our model sheds new light on how P. falciparum binds within brain microvessels and provides a powerful method for future investigations of recruitment of human brain pathogens to the blood vessel lining of the brain.

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel.

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Human Organ-Specific Endothelial Cell Heterogeneity.

The endothelium first forms in the blood islands in the extra-embryonic yolk sac and then throughout the embryo to establish circulatory networks that further acquire organ-specific properties during development to support diverse organ functions. Here, we investigated the properties of endothelial cells (ECs), isolated from four human major organs-the heart, lung, liver, and kidneys-in individual fetal tissues at three months' gestation, at gene expression, and at cellular function levels. We showed that organ-specific ECs have distinct expression patterns of gene clusters, which support their specific organ development and functions. These ECs displayed distinct barrier properties, angiogenic potential, and metabolic rate and support specific organ functions. Our findings showed the link between human EC heterogeneity and organ development and can be exploited therapeutically to contribute in organ regeneration, disease modeling, as well as guiding differentiation of tissue-specific ECs from human pluripotent stem cells.

Decellularized Human Kidney Cortex Hydrogels Enhance Kidney Microvascular Endothelial Cell Maturation and Quiescence.

The kidney peritubular microvasculature is highly susceptible to injury from drugs and toxins, often resulting in acute kidney injury and progressive chronic kidney disease. Little is known about the process of injury and regeneration of human kidney microvasculature, resulting from the lack of appropriate kidney microvascular models that can incorporate the proper cells, extracellular matrices (ECMs), and architectures needed to understand the response and contribution of individual vascular components in these processes. In this study, we present methods to recreate the human kidney ECM (kECM) microenvironment by fabricating kECM hydrogels derived from decellularized human kidney cortex. The majority of native matrix proteins, such as collagen-IV, laminin, and heparan sulfate proteoglycan, and their isoforms were preserved in similar proportions as found in normal kidneys. Human kidney peritubular microvascular endothelial cells (HKMECs) became more quiescent when cultured on this kECM gel compared with culture on collagen-I-assessed using phenotypic, genotypic, and functional assays; whereas human umbilical vein endothelial cells became stimulated on kECM gels. We demonstrate for the first time that human kidney cortex can form a hydrogel suitable for use in flow-directed microphysiological systems. Our findings strongly suggest that selecting the proper ECM is a critical consideration in the development of vascularized organs on a chip and carries important implications for tissue engineering of all vascularized organs.

Ultrasound-guided photoacoustic imaging-directed re-endothelialization of acellular vasculature leads to improved vascular performance.

As increasing effort is dedicated to investigating the regenerative capacity of decellularized tissues, research has progressed to recellularizing these tissues prior to implantation. The delivery and support of cells seeded throughout acellular scaffolds are typically conducted through the vascular axis of the tissues. However, it is unclear how cell concentration and injection frequency can affect the distribution of cells throughout the scaffold. Furthermore, what effects re-endothelialization have on vascular patency and function are not well understood. We investigated the use of ultrasound-guided photoacoustic (US/PA) imaging as a technique to visualize the distribution of microvascular endothelial cells within an optimized acellular construct upon re-endothelialization and perfusion conditioning. We also evaluated the vascular performance of the re-endothelialized scaffold using quantitative vascular corrosion casting (qVCC) and whole-blood perfusion. We found US/PA imaging was an effective technique to visualize the distribution of cells. Cellular retention following perfusion conditioning was also detected with US/PA imaging. Finally, we demonstrated that a partial recovery of vascular performance is possible following re-endothelialization-confirmed by fewer extravasations in qVCC and improved blood clearance following whole-blood perfusion. STATEMENT OF SIGNIFICANCE: Re-endothelialization is a method that enables decellularized tissue to become useful as a tissue engineering construct by creating a nutrient delivery and waste removal system for the entire construct. Our approach utilizes a decellularization method that retains the basement ECM of a highly vascularized tissue upon which endothelial cells can be injected to form an endothelium. The US/PA method allows for rapid visualization of cells within a construct several cm thick. This approach can be experimentally used to observe changes in cellular distribution over large intervals of time, to help optimize cell seeding parameters, and to verify cell retention within re-endothelialized constructs. This approach has temporal and depth advantages compared to section reconstruction and imaged fluorophores respectively.

A Novel Three-Dimensional Human Peritubular Microvascular System.

Human kidney peritubular capillaries are particularly susceptible to injury, resulting in dysregulated angiogenesis, capillary rarefaction and regression, and progressive loss of kidney function. However, little is known about the structure and function of human kidney microvasculature. Here, we isolated, purified, and characterized human kidney peritubular microvascular endothelial cells (HKMECs) and reconstituted a three-dimensional human kidney microvasculature in a flow-directed microphysiologic system. By combining epithelial cell depletion and cell culture in media with high concentrations of vascular endothelial growth factor, we obtained HKMECs of high purity in large quantity. Unlike other endothelial cells, isolated HKMECs depended on high vascular endothelial growth factor concentration for survival and growth and exhibited high tubulogenic but low angiogenic potential. Furthermore, HKMECs had a different transcriptional profile. Under flow, HKMECs formed a thin fenestrated endothelium with a functional permeability barrier. In conclusion, this three-dimensional HKMEC-specific microphysiologic system recapitulates human kidney microvascular structure and function and shows phenotypic characteristics different from those of other microvascular endothelial cells.

Preservation of Capillary-beds in Rat Lung Tissue Using Optimized Chemical Decellularization.

Promoting regeneration using scaffolds created by decellularizing native tissue is becoming a popular technique applied to a variety of tissues. We demonstrate a method to decellularize highly vascular tissue keeping the vascular structure intact down to the capillary scale. Using vascular corrosion casting (VCC), we created a method for quantitatively assessing the functionality of vascular extracellular matrix (ECM) following decellularization. Murine lung tissue was decellularized using a number of techniques, then characterized using standard histological methods, as well as our quantitative VCC (qVCC) technique. Using an optimized acellular method, we successfully decellularized lung tissue while leaving behind a patent vascular network based on qualitative and quantitative histological methods.

Functional characterization of optimized acellular peripheral nerve graft in a rat sciatic nerve injury model.

OBJECTIVES: Acellular grafts are a viable option for use in nerve reconstruction surgeries. Recently, our lab created a novel optimized decellularization procedure that removes immunological material while leaving the majority of the extracellular matrix structure intact. The optimized acellular (OA) graft has been shown to elicit an immune response equal to or less than that elicited by the isograft, the analog of the autograft in the rat model. We investigated the performance of the OA graft to provide functional recovery in a long-term study. METHODS: We performed a long-term functional regeneration evaluation study using the sciatic functional index to quantify recovery of Lewis rats at regular time intervals for up to 52 weeks after graft implantation following 1 cm sciatic nerve resection. OA grafts were compared against other decellularized methods (Sondell treatment and thermal decellularization), as well as the isograft and primary neurorrhaphy. RESULTS: The OA graft supported comparable functional recovery to the isograft and superior regeneration to thermal and Sondell decellularization methods. Furthermore, the OA graft promoted early recovery to a greater degree compared to acellular grafts obtained using either the thermal or the Sondell methods. DISCUSSION: Equivalent functional recovery to the isograft suggests that the OA nerve graft may be a future clinical alternative to the current autologous tissue graft.