Dissection of heterocellular cross-talk in vascularized cardiac tissue mimetics.

Cellular specialization and interaction with other cell types in cardiac tissue is essential for the coordinated function of cell populations in the heart. The complex interplay between cardiomyocytes, endothelial cells and fibroblasts is necessary for adaptation but can also lead to pathophysiological remodeling. To understand this complex interplay, we developed 3D vascularized cardiac tissue mimetics (CTM) to study heterocellular cross-talk in hypertrophic, hypoxic and fibrogenic environments. This 3D platform responds to physiologic and pathologic stressors and mimics the microenvironment of diseased tissue. In combination with endothelial cell fluorescence reporters, these cardiac tissue mimetics can be used to precisely visualize and quantify cellular and functional responses upon stress stimulation. Utilizing this platform, we demonstrate that stimulation of α/ß-adrenergic receptors with phenylephrine (PE) promotes cardiomyocyte hypertrophy, metabolic maturation and vascularization of CTMs. Increased vascularization was promoted by conditioned medium of PE-stimulated cardiomyocytes and blocked by inhibiting VEGF or upon ß-adrenergic receptor antagonist treatment, demonstrating cardiomyocyte-endothelial cross-talk. Pathophysiological stressors such as severe hypoxia reduced angiogenic sprouting and increased cell death, while TGF ß2 stimulation increased collagen deposition concomitant to endothelial-to-mesenchymal transition. In sum, we have developed a cardiac 3D culture system that reflects native cardiac tissue function, metabolism and morphology - and for the first time enables the tracking and analysis of cardiac vascularization dynamics in physiology and pathology.

Endothelial cell-cardiomyocyte crosstalk in heart development and disease.

The crosstalk between endothelial cells and cardiomyocytes has emerged as a requisite for normal cardiac development, but also a key pathogenic player during the onset and progression of cardiac disease. Endothelial cells and cardiomyocytes are in close proximity and communicate through the secretion of paracrine signals, as well as through direct cell-to-cell contact. Here, we provide an overview of the endothelial cell-cardiomyocyte interactions controlling heart development and the main processes affecting the heart in normal and pathological conditions, including ischaemia, remodelling and metabolic dysfunction. We also discuss the possible role of these interactions in cardiac regeneration and encourage the further improvement of in vitro models able to reproduce the complex environment of the cardiac tissue, in order to better define the mechanisms by which endothelial cells and cardiomyocytes interact with a final aim of developing novel therapeutic opportunities.

Monocytes enhance neutrophil-induced blister formation in an ex vivo model of bullous pemphigoid.

BACKGROUND: Lesions of bullous pemphigoid (BP), an autoimmune subepidermal blistering disease characterized by the presence of tissue-bound and circulating autoantibodies to hemidesmosomal antigens, harbor a mixed inflammatory cellular infiltrate. In various models, neutrophils, eosinophils, mast cells, monocytes as well as B and T cells have been shown to be involved in the pathogenesis of BP. However, their interactions with and effective role in blister formation remain uncertain. This study was aimed at investigating the effect of monocyte/neutrophil interaction on blister formation in an ex vivo BP model. METHODS: Skin cryosections were incubated with purified human neutrophils and monocytes, in the presence or absence of BP autoantibodies. Production of reactive oxygen species (ROS), degranulation, mediator release (neutrophil elastase [NE], myeloperoxidase [MPO], matrix metalloproteinase-9 [MMP-9]), binding of Fcγ receptor (CD16, CD32, CD64), and cell adhesion (CD18, ICAM-1) was investigated using appropriate inhibitors. Dermal-epidermal separation (DES) was assessed by light microscopy and quantified by Fiji software. RESULTS: Monocytes and neutrophils synergistically interact resulting in a significantly higher DES compared to either monocytes or neutrophils separately (P < .0001). Monocyte/neutrophil-induced DES was associated with increased ROS production and was dependent on adhesion and FcγRIII binding. Upon stimulation by the granule-poor fraction of monocyte supernatants, neutrophils increased their release of MMP-9, thereby also DES at the dermal-epidermal junction of skin cryosections. CONCLUSION: Our observations suggest that the interaction of cells, as shown here for monocytes and neutrophils, enhances mediator release resulting in an increased subepidermal blister formation. Thus, blocking intercellular cross talk promises a new therapeutic approach for blocking tissue damage in BP.

Measurement of extracellular vesicles as biomarkers of consequences or cause complications of pathological states, and prognosis of both evolution and therapeutic safety/efficacy.

Utility of EVs, as biomarkers of cause or consequence of various pathological complications, and prognosis of blood components' therapy in terms of safety/efficacy and their potential associated hazards, primed by EVs involvements in pro-inflammatory, immunomodulatory and activations of both pro/anti-coagulatory and others associated pathways, as well as various cellular cross talks, are highlighted as the fundamental. Today EVs are becoming the "buzz" words of the current diagnosis, development and research [DDR] strategies, with the aim of ensuring safer therapeutic approaches in the current clinical practices, also incorporating their potential in long term cost effectiveness in health care systems. The main focus of this manuscript is to review the current opinions in some fundamental areas of EVs involvements in health and diseases. Firstly, our goal is highlighting what are EVs/MVs/MPs and how are they generated in physiology, pathology or blood products; classification and significance of EVs generated in vivo; followed by consequences and physiological/pathological induced effects of EVs generation in vivo. Secondly, specific cell origin EVs and association with malignancy; focus on EVs carrying TF and annexin V as a protective protein for harmful effects of EVs, and associations with LA; and incidence of anti-annexin V antibodies are also discussed. Thirdly, utility of EVs is presented: as diagnostic tools of disease markers; prognosis and follow-up of clinical states; evaluation of therapy efficacy; quality and risk assessment of blood products; followed by the laboratory tools for exploring, characterizing and measuring EVs, and/or their associated activity, using our own experiences of capture based assays. Finally, in perspective, the upcoming low volume sampling, fast, reliable and reproducibility and friendly use laboratory tools and the standardization of measurement methods are highlighted with the beneficial effects that we are witnessing in both wound healing and tissue remodeling, with an expected blockbuster status EVs as future therapeutic directions.

Assembled/Disassembled Modular Scaffolds for Multicellular Tissue Engineering.

The behavior of tissue resident cells can be influenced by the spatial arrangement of cellular interactions. Therefore, it is of significance to precisely control the spatial organization of various cells within multicellular constructs. It remains challenging to construct a versatile multicellular scaffold with ordered spatial organization of multiple cell types. Herein, a modular multicellular tissue engineering scaffold with ordered spatial distribution of different cell types is constructed by assembling varying cell-laden modules. Interestingly, the modular scaffolds can be disassembled into individual modules to evaluate the specific contribution of each cell type in the system. Through assembling cell-laden modules, the macrophage-mesenchymal stem cell (MSC), endothelial cell-MSC, and chondrocyte-MSC co-culture models are successfully established. The in vitro results indicate that the intercellular cross-talk can promote the proliferation and differentiation of each cell type in the system. Moreover, MSCs in the modular scaffolds may regulate the behavior of chondrocytes through the nuclear factor of activated T-Cells (NFAT) signaling pathway. Furthermore, the modular scaffolds loaded with co-cultured chondrocyte-MSC exhibit enhanced regeneration ability of osteochondral tissue, compared with other groups. Overall, this work offers a promising strategy to construct a multicellular tissue engineering scaffold for the systematic investigation of intercellular cross-talk and complex tissue engineering.