multimodal-approach-to-assess-bone-regeneration-scaffold

Multimodal Approach to Assess Bone Regeneration and Scaffold Performance

The clinical success of an implant largely depends on its ability to promote bone repair and facilitate osteointegration. While significant advancements ...

Federal University of Rio de Janeiro

The therapeutic potential of extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) in kidney injury has been largely reported. However, new approaches are necessary to optimize the efficacy in the treatment of renal diseases. MSCs physiologically are under a low O partial pressure (pO), and culturing adipose-derived MSCs (ADMSCs) in hypoxia alters their secretory paracrine properties. The aim of this study was to evaluate whether hypoxia preconditioning of ADMSCs alters the properties of secreted EVs to improve renal recovery after ischemia-reperfusion injury (IRI).

Adipose-Derived Mesenchymal Stromal Cells Under Hypoxia: Changes in Extracellular Vesicles Secretion and Improvement of Renal Recovery after Ischemic Injury.

Malaria is a major parasitic disease of humans and is a health public problem that affects more than 100 countries. In 2017, it caused nearly half a million deaths out of 219 million infections. Malaria is caused by the protozoan parasites of the genus Plasmodium and is transmitted by female mosquitoes of the genus Anopheles. Once in the bloodstream, Plasmodium merozoites invade erythrocytes and proliferate until the cells lyses and release new parasites that invade other erythrocytes. Remarkably, they can manipulate the vertebrate host's lipid metabolism pathways, since they cannot synthesize lipid classes that are essential for their development and replication. In this study, we show that mice infected with Plasmodium chabaudi present a completely different plasma profile from control mice, with marked hyperproteinemia, hypertriglyceridemia, hypoglycemia, and hypocholesterolemia. In addition, white adipose and hepatic tissue and analyses from infected animals revealed the accumulation of triacylglycerol in both tissues and free fatty acids and free cholesterol in the liver. Hepatic mRNA and protein expression of key enzymes and transcription factors involved in lipid metabolism were also altered by P. chabaudi infection, leading to a lipogenic state. The enzyme 5' AMP-activated protein kinase (AMPK), a master regulator of cell energetic metabolism, was also modulated by the parasite, which reduced AMPK phosphorylation levels upon infection. Pretreatment with metformin for 21 days followed by infection with P. chabaudi was effective in preventing infection of mice and also lowered the hepatic accumulation of lipids while activating AMPK. Together, these results provide new and important information on the specific molecular mechanisms induced by the malaria parasite to regulate hepatic lipid metabolism in order to facilitate its development, proliferation, and lifespan in its vertebrate host.

Plasmodium Infection Induces Dyslipidemia and a Hepatic Lipogenic State in the Host through the Inhibition of the AMPK-ACC Pathway.

Induced pluripotent stem cells (iPSC) have been the focus of several studies due to their wide range of application, including in cellular therapy. The use of iPSC in regenerative medicine is limited by their tumorigenic potential. Extracellular vesicles (EV) derived from stem cells have been shown to support renal recovery after injury. However, no investigation has explored the potential of iPSC-EV in the treatment of kidney diseases. To evaluate this potential, we submitted renal tubule cells to hypoxia-reoxygenation injury, and we analyzed cell death rate and changes in functional mitochondria mass. An in vivo model of ischemia-reperfusion injury was used to evaluate morphological and functional alterations. Gene array profile was applied to investigate the mechanism involved in iPSC-EV effects. In addition, EV derived from adipose mesenchymal cells (ASC-EV) were also used to compare the potential of iPSC-EV in support of tissue recovery. The results showed that iPSC-EV were capable of reducing cell death and inflammatory response with similar efficacy than ASC-EV. Moreover, iPSC-EV protected functional mitochondria and regulated several genes associated with oxidative stress. Taken together, these results show that iPSC can be an alternative source of EV in the treatment of different aspects of kidney disease.

Extracellular Vesicles Derived from Induced Pluripotent Stem Cells Promote Renoprotection in Acute Kidney Injury Model.

Acute kidney injury (AKI) caused by ischemia followed by reperfusion (I/R) is characterized by intense anion superoxide (O) production and oxidative damage. We investigated whether extracellular vesicles secreted by adipose tissue mesenchymal cells (EVs) administered during reperfusion can suppress the exacerbated mitochondrial O formation after I/R. We used Wistar rats subjected to bilateral renal arterial clamping (30 min) followed by 24 h of reperfusion. The animals received EVs (I/R + EVs group) or saline (I/R group) in the kidney subcapsular space. The third group consisted of false-operated rats (SHAM). Mitochondria were isolated from proximal tubule cells and used immediately. Amplex Red™ was used to measure mitochondrial O formation and MitoTracker™ Orange to evaluate inner mitochondrial membrane potential (Δψ). In vitro studies were carried out on human renal proximal tubular cells (HK-2) co-cultured or not with EVs under hypoxic conditions. Administration of EVs restored O formation to SHAM levels in all mitochondrial functional conditions. The gene expression of  and  remained unmodified; transcription of  () was upregulated. The co-cultures of HK-2 cells with EVs revealed an intense decrease in apoptosis. We conclude that the mechanisms by which EVs favor long-term recovery of renal structures and functions after I/R rely on a decrease of mitochondrial O formation with the aid of the upregulated antioxidant HO-1/Nuclear factor erythroid 2-related factor 2 system, thus opening new vistas for the treatment of AKI.

Early Effects of Extracellular Vesicles Secreted by Adipose Tissue Mesenchymal Cells in Renal Ischemia Followed by Reperfusion: Mechanisms Rely on a Decrease in Mitochondrial Anion Superoxide Production.

Extracellular vesicles (EVs) are known as molecular carriers involved in cell communication and the regulation of (patho)physiological processes. miRNAs and growth factors are the main contents of EVs which make them a good candidate for the treatment of diseases caused by ischemia, but the low production of EVs by a cell producer and a significant variation of the molecular contents in EVs according to the cell source are the main limitations of their widespread use. Here, we show how to improve the therapeutic properties of mesenchymal stromal cell (MSC)-derived EVs (MSC-EVs) by modifying MSCs to enrich these EVs with specific angiomiRs (miR-135b or miR-210) using lentiviral vectors carrying miR-135b or miR-210. MSCs were obtained from the mouse bone marrow and transduced with a corresponding lentivector to overexpress miR-135b or miR-210. The EVs were then isolated by ultracentrifugation and characterized using a flow cytometer and a nanoparticle tracking analyzer. The levels of 20 genes in the MSCs and 12 microRNAs in both MSCs and EVs were assessed by RT‒qPCR. The proangiogenic activity of EVs was subsequently assessed in human umbilical vein endothelial cells (HUVECs). The results confirmed the overexpression of the respective microRNA in modified MSCs. Moreover, miR-135b overexpression upregulated miR-210-5p and follistatin, whereas the overexpression of miR-210 downregulated miR-221 and upregulated miR-296. The tube formation assay showed that EVs from MSCs overexpressing miR-210-5p (EVmiR210) significantly promoted tubular structure formation in HUVECs. A significant increase in angiogenic proteins (PGF, endothelin 1, and artemin) and genes (VEGF, activin A, and IGFBP1) in HUVECs treated with VEmiR210 justifies the better tubular structure formation of these cells compared with that of EVmiR135b-treated HUVECs, which showed upregulated expression of only artemin. Collectively, our results show that the EV cargo can be modified by lentiviral vectors to enrich specific miRNAs to achieve a specific angiogenic potential.

Overexpression of mir-135b and mir-210 in mesenchymal stromal cells for the enrichment of extracellular vesicles with angiogenic factors.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic disorder that causes accelerated aging and a high risk of cardiovascular complications. However, the underlying mechanisms of cardiac complications of this syndrome are not fully understood. This study modeled HGPS using cardiomyocytes (CM) derived from induced pluripotent stem cells (iPSC) derived from a patient with HGPS and characterized the biophysical, morphological, and molecular changes found in these CM compared to CM derived from a healthy donor. Electrophysiological recordings suggest that the HGPS-CM was functional and had normal electrophysiological properties. Electron tomography showed nuclear morphology alteration, and the 3D reconstruction of electron tomography images suggests structural abnormalities in HGPS-CM mitochondria, however, there was no difference in mitochondrial content as measured by Mitotracker. Immunofluorescence indicates nuclear morphological alteration and confirms the presence of Troponin T. Telomere length was measured using qRT-PCR, and no difference was found in the CM from HGPS when compared to the control. Proteomic analysis was carried out in a high-resolution system using Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). The proteomics data show distinct group separations and protein expression differences between HGPS and control-CM, highlighting changes in ribosomal, TCA cycle, and amino acid biosynthesis, among other modifications. Our findings show that iPSC-derived cardiomyocytes from a Progeria Syndrome patient have significant changes in mitochondrial morphology and protein expression, implying novel mechanisms underlying premature cardiac aging.

Modelling premature cardiac aging with induced pluripotent stem cells from a hutchinson-gilford Progeria Syndrome patient.

Malaria remains a major global threat, representing a severe public health problem worldwide. Annually, it is responsible for a high rate of morbidity and mortality in many tropical developing countries where the disease is endemic. The causative agent of malaria, Plasmodium spp., exhibits a complex life cycle, alternating between an invertebrate vector, which transmits the disease, and the vertebrate host. The disease pathology observed in the vertebrate host is attributed to the asexual development of Plasmodium spp. inside the erythrocyte. Once inside the red blood cell, malaria parasites cause extensive changes in the host cell, increasing membrane rigidity and altering its normal discoid shape. Additionally, during their intraerythrocytic development, malaria parasites incorporate and degrade up to 70 % of host cell hemoglobin. This mechanism is essential for parasite development and represents an important drug target. Blocking the steps related to hemoglobin endocytosis or degradation impairs parasite development and can lead to its death. The ultrastructural analysis of hemoglobin endocytosis on Plasmodium spp. has been broadly explored along the years. However, it is only recently that the proteins involved in this process have started to emerge. Here, we will review the most important features related to hemoglobin endocytosis and catabolism on malaria parasites. A special focus will be given to the recent analysis obtained through 3D visualization approaches and to the molecules involved in these mechanisms.

Endocytosis in malaria parasites: An ultrastructural perspective of membrane interplay in a unique infection model.

This study investigated whether chronic undernutrition alters the mitochondrial structure and function in renal proximal tubule cells, thus impairing fluid transport and homeostasis. We previously showed that chronic undernutrition downregulates the renal proximal tubules (Na+K)ATPase, the main molecular machine responsible for fluid transport and ATP consumption. Male rats received a multifactorial deficient diet, the so-called Regional Basic Diet (RBD), mimicking those used in impoverished regions worldwide, from weaning to a juvenile age (3 months). The diet has a low content (8 %) of poor-quality proteins, low lipids, and no vitamins compared to control (CTR). We investigated citrate synthase activity, mitochondrial respiration (oxygraphy) in phosphorylating and non-phosphorylating conditions with different substrates/inhibitors, potential across the internal membrane (Δψ), and anion superoxide/HO formation. The data were correlated with ultrastructural alterations evaluated using transmission electron microscopy (TEM) and focused ion beam scanning electron microscopy (FIB-SEM). Citrate synthase activity decreased (∼50 %) in RBD rats, accompanied by a similar reduction in respiration in non-phosphorylating conditions, maximum respiratory capacity, and ATP synthesis. The Δψ generation and its dissipation after carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone remained unmodified in the survival mitochondria. HO production increased (∼100 %) after Complex II energization. TEM demonstrated intense matrix vacuolization and disruption of cristae junctions in a subpopulation of RBD mitochondria, which was also demonstrated in the 3D analysis of FIB-SEM tomography. In conclusion, chronic undernutrition impairs mitochondrial functions in renal proximal tubules, with profound alterations in the matrix and internal membrane ultrastructure that culminate with the compromise of ATP supply for transport processes.

Chronic undernutrition impairs renal mitochondrial respiration accompanied by intense ultrastructural damage in juvenile rats.

We investigated the biomineralization process of calcium carbonate deposition in the spicules of the calcareous sponge Heteropia glomerosa (Porifera, Calcarea). The finely polished spicules, composed of Mg-calcite, present a pattern of concentric lines spaced 400 nm apart when observed by scanning electron microscopy. We showed by electron backscattered diffraction that the whole spicule length has the same crystallographic orientation. Still, misorientation of up to 1.8° in adjacent regions (∼ 2 µm) and a continuous increase in the misalignment of up to 4.5° in regions separated by 300 µm were present. The sponge cells (mainly sclerocytes and pinacocytes) near the mineralization zone contain a high number of vesicles rich in calcium, which could be involved in the spicule biomineralization. We showed by electron and ion microscopies that the spicule growth occurs through the addition calcium carbonate granules, which form near the membrane of the sclerocyte, the cell responsible for biomineralization. The granules were deposited layer by layer on the surface of the spicule, increasing the biomineral thickness. Domains of 1-3 µm containing facets partially connected and surrounded by organic material were observed in an intermediate stage of the spicule growth. Misorientation between these domains was approximately 2°, similar to the misorientation obtained by electron backscattered diffraction, indicating that the spicule is formed by the addition of granules fusing in a predominant orientation.

Article	Year
Adipose-Derived Mesenchymal Stromal Cells Under Hypoxia: Changes in Extracellular Vesicles Secretion and Improvement of Renal Recovery after Ischemic Injury. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology\| PubMed ID: 31099507	2019
Plasmodium Infection Induces Dyslipidemia and a Hepatic Lipogenic State in the Host through the Inhibition of the AMPK-ACC Pathway. Scientific reports\| PubMed ID: 31604978	2019
Extracellular Vesicles Derived from Induced Pluripotent Stem Cells Promote Renoprotection in Acute Kidney Injury Model. Cells\| PubMed ID: 32079274	2020
Early Effects of Extracellular Vesicles Secreted by Adipose Tissue Mesenchymal Cells in Renal Ischemia Followed by Reperfusion: Mechanisms Rely on a Decrease in Mitochondrial Anion Superoxid... International journal of molecular sciences\| PubMed ID: 35328327	2022
Overexpression of mir-135b and mir-210 in mesenchymal stromal cells for the enrichment of extracellular vesicles with angiogenic factors. PloS one\| PubMed ID: 35972944	2022
Modelling premature cardiac aging with induced pluripotent stem cells from a hutchinson-gilford Progeria Syndrome patient. Frontiers in physiology\| PubMed ID: 36505085	2022
Endocytosis in malaria parasites: An ultrastructural perspective of membrane interplay in a unique infection model. Current topics in membranes\| PubMed ID: 39181577	2024
Chronic undernutrition impairs renal mitochondrial respiration accompanied by intense ultrastructural damage in juvenile rats. Biochemical and biophysical research communications\| PubMed ID: 39182354	2024
Calcium carbonate deposition in the spicules of the sponge Heteropia glomerosa (Porifera, Calcarea). Journal of structural biology\| PubMed ID: 40378937	2025

Camila Wendt

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