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.
BackgroundThe marrow microenvironment and vasculature plays a critical role in regulating hematopoietic cell recruitment, residence, and maturation. Extensive in vitro and in vivo studies have aimed to understand the marrow cell types that contribute to hematopoiesis and the stem cell environment. Nonetheless, in vitro models are limited by a lack of complex multicellular interactions, and cellular interactions are not easily manipulated in vivo. Here, we develop an engineered human vascular marrow niche to examine the three-dimensional cell interactions that direct hematopoietic cell trafficking.MethodsUsing soft lithography and injection molding techniques, fully endothelialized vascular networks were fabricated in type I collagen matrix, and co-cultured under flow with embedded marrow fibroblast cells in the matrix. Marrow fibroblast (mesenchymal stem cells (MSCs), HS27a, or HS5) interactions with the endothelium were imaged via confocal microscopy and altered endothelial gene expression was analyzed with RT-PCR. Monocytes, hematopoietic progenitor cells, and leukemic cells were perfused through the network and their adhesion and migration was evaluated.ResultsHS27a cells and MSCs interact directly with the vessel wall more than HS5 cells, which are not seen to make contact with the endothelial cells. In both HS27a and HS5 co-cultures, endothelial expression of junctional markers was reduced. HS27a co-cultures promote perfused monocytes to adhere and migrate within the vessel network. Hematopoietic progenitors rely on monocyte-fibroblast crosstalk to facilitate preferential recruitment within HS27a co-cultured vessels. In contrast, leukemic cells sense fibroblast differences and are recruited preferentially to HS5 and HS27a co-cultures, but monocytes are able to block this sensitivity.ConclusionsWe demonstrate the use of a microvascular platform that incorporates a tunable, multicellular composition to examine differences in hematopoietic cell trafficking. Differential recruitment of hematopoietic cell types to distinct fibroblast microenvironments highlights the complexity of cell-cell interactions within the marrow. This system allows for step-wise incorporation of cellular components to reveal the dynamic spatial and temporal interactions between endothelial cells, marrow-derived fibroblasts, and hematopoietic cells that comprise the marrow vascular niche. Furthermore, this platform has potential for use in testing therapeutics and personalized medicine in both normal and disease contexts.Electronic supplementary materialThe online version of this article (10.1186/s13287-018-0808-2) contains supplementary material, which is available to authorized users.
Rationale The mitochondrial permeability transition pore (mPTP) is a well-known initiator of cell death that is increasingly recognized as a physiological modulator of cellular metabolism. Objective We sought to identify how the genetic deletion of a key regulatory subunit of the mPTP, Cyclophilin D (CypD), influenced endothelial metabolism and intracellular signaling. Methods and Results In cultured primary human endothelial cells (ECs), genetic targeting of CypD using siRNA or shRNA resulted in a constitutive increase in mitochondrial matrix Ca2+ and NADH. Elevated matrix NADH in turn diminished the cytosolic NAD+/NADH ratio and triggered a subsequent downregulation of the NAD+-dependent deacetylase SIRT1. Downstream of SIRT1, CypD-deficient ECs exhibited reduced PTEN expression and a constitutive rise in the phosphorylation of angiogenic Akt. Similar changes in SIRT1, PTEN, and Akt were also noted in the aorta and lungs of CypD KO mice. Functionally, CypD-deficient ECs and aortic tissue from CypD KO mice exhibited a dramatic increase in angiogenesis at baseline and when exposed to vascular endothelial growth factor (VEGF). The NAD+ precursor Nicotinamide mononucleotide restored the cellular NAD+/NADH ratio and normalized the CypD-deficient phenotype. CypD KO mice also presented accelerated wound healing and increased neovascularization upon tissue injury as monitored by optical microangiography (OMAG). Conclusion Our study reveals the importance of the mPTP in the regulation of endothelial mitochondrial metabolism and vascular function. The mitochondrial regulation of SIRT1 has broad implications in the epigenetic regulation of endothelial phenotype.
Ischaemic heart disease represents the leading cause of death worldwide. Heart failure following myocardial infarction (MI) is associated with severe fibrosis formation and cardiac remodeling. Recently, injectable hydrogels have emerged as a promising approach to repair the infarcted heart and improve heart function through minimally invasive administration. Here, we developed a novel injectable human amniotic membrane (hAM) matrix to enhance cardiac regeneration following MI. Human amniotic membrane was isolated from human placenta and engineered to be a thermo-responsive, injectable gel around body temperature. Ultrasoundguided injection of hAM matrix into the rat MI hearts significantly improved cardiac contractility, as measured by ejection fraction (EF), and decreased fibrosis. Our results demonstrate the feasibility of engineering an injectable hAM matrix and its efficacy in attenuating degenerative changes in cardiac function following MI, which may have broad applications in tissue regeneration.
Vasculature is an interface between the circulation and the hematopoietic tissue providing the means for hundreds of billions of blood cells to enter the circulation every day in a regulated fashion. The precise mechanisms that control the interactions of hematopoietic cells with the vessel wall are largely undefined. Here, we report on the development of an in vitro 3D human marrow vascular microenvironment (VME) to study hematopoietic trafficking and the release of blood cells, specifically platelets. We show that mature megakaryocytes from aspirated marrow as well as megakaryocytes differentiated in culture from CD34+ cells can be embedded in a collagen matrix containing engineered microvessels to create a thrombopoietic VME. These megakaryocytes continue to mature, penetrate the vessel wall, and release platelets into the vessel lumen. This process can be blocked with the addition of antibodies specific for CXCR4, indicating that CXCR4 is required for megakaryocyte migration, though whether it is sufficient is unclear. The 3D marrow VME system shows considerable potential for mechanistic studies defining the role of marrow vasculature in thrombopoiesis. Through a stepwise addition or removal of individual marrow components, this model provides potential to define key pathways responsible for the release of platelets and other blood cells.
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