Leader: Gabriella Minchiotti (CNR); Other collaborator(s): Ombretta Guardiola (CNR), Angela Gambardella (CNR), Sandro De Falco (CNR), Valeria Tarallo (CNR), Antonio Adinolfi (CNR), Fernando Gianfrancesco (CNR), Sharon Russo (CNR)
We will analyze mechanisms of tissue remodeling/fibrosis in different organs and tissues including eye, bone marrow and skeletal muscle, and we will aim to integrate results into a predictive model of onset and severity of diseases. We will combine mouse models, imaging, trascriptomic analysis, and machine learning. Specifically, we will study:
Brief description of the activities and of the intermediate results: We have started to analyze the difference in the development of subretinal fibrosis in C57Bl6 wild type mice and PlGF-DE-Kin mice. Subretinal fibrosis has been established in these mice using the model of laser-induced choroidal neovascularization. In this model, CNV lesions will be observed by Isolectin B4 staining, while subretinal fibrosis will be stained by Collagen I. Eyes will be analyzed after 7, 21 e 35 day from laser damage.At same time points, total RNA will be harvested from RPE/choroid tissues in order to confirm the presence of endothelial to mesenchymal transition, which has been shown to have a pivotal role in the development of subretinal fibrosis.
To investigate the composition of bone marrow-derived hematopoietic lineage in the Zfp687 knock-out and Pfn1c.318_321del knock-in mouse models, we focused on osteoclast progenitor cells, which are part of the hematopoietic lineage. To this end, we assessed the abundance of osteoclast progenitors by flow cytometry, using specific surface markers: Ter119-B220-CD117+CD115+CD11b-. We observed a significant reduction in the percentage of osteoclast progenitors in the Zfp687-KO bone marrow-derived cells. Conversely, in the Pfn1-KI model bone marrow, we noted an increase in these cells. These findings suggest that Zfp687 and Pfn1 play a role in the reservoir of hematopoietic-derived osteoclast precursors.
We have further assessed the molecular and functional features of activated muscle stem cells (MuSCs) isolated from injured skeletal muscles that express different levels of CRIPTO at the cell membrane. RNA-seq exploration identified key canonical pathways that are deregulated in CRIPTOPos and CRIPTONeg cell fractions. These include G protein-coupled receptor (GPR) and calcium signaling pathways, which have been poorly explored so far in the context of MuSC heterogeneity and their adaptive responses. We have demoinstrated that CRIPTO micro-heterogeneity is generated and maintained in the MuSCs population through a process of intracellular trafficking coupled with active shedding of CRIPTO from the plasma membrane.
To characterize the impact of myeloid-specific CRIPTO KO on different macrophage subpopulations and other cellular compartments, including MuScs, ECs and FAPS in skeletal muscle regeneration, scRNA-Seq was performed in TA muscles of control and Cripto KO mice at days 3 and 5 post-injury. The analysis is currently ongoing.
We also explored a gain-of-function approach using recombinant soluble CRIPTO protein (sCRIPTO) as a pharmacological strategy to mitigate EndoMT and enhance skeletal muscle regeneration. Intramuscular administration of sCRIPTO restored proper accumulation of CD206+ anti-inflammatory macrophages and restricted EndoMT in CRIPTO LOF mice.
In collaboration with Dr. Dror Seliktar at the Technion Institute, we developed a PEG-fibrinogen microsphere delivery system for localized, sustained release of sCRIPTO protein toward the accelerated repair of damaged muscle tissue following acute injuries.
In order to study subretinal fibrosis, we performed laser-induced choroidal neovascularization (CNV) lesions in C57Bl6 wild type and PlGF-DE-Kin mice. We observed that CNV size (stained with Isolectin B4) reached a maximum on day 7 and then regressed at 21 days and completely disappeared by day 35. During the same period, subretinal fibrosis (stained with Collagen I) progressively increased, reaching a peak at day 35. We confirmed significant reduction of CNV size and we observed a severe impairment of subretinal fibrosis in PlGF-DE mice compared to control. Expression Analysis of EndMT markers and bulk RNAseq on isolated mouse choroidal endothelial cells (mCECs) from C57Bl6 wild type and PlGF-DE-Kin mice are in progress to generate an in vitro system to study EndEMT.
To investigate the transcriptional networks involving Zfp687 and their influence on target genes, which lead to a reduction in the percentage of osteoclast progenitors in Zfp687-KO bone marrow-derived cells, we sorted cells using flow cytometry (Ter119-B220-CD117+CD115+CD11b-) and extracted RNA to analyze their transcriptomic profile. The analysis of this experiment is currently ongoing.
To further uncover the molecular dynamics of various cell types, delineate cell differentiation pathways, and identify cell fate changes during hematopoietic lineage decisions following alterations in the Zfp687 gene, we are performing single-cell RNA sequencing (scRNA-seq) on bone marrow-derived HSCs/HPSCs. These cells are stained with fluorochrome-conjugated antibodies against the surface markers Ter119, B220, and CD117 to identify the Ter119-B220-CD117+ population.
In order to study and identify new gene deregulated during subretinal fibrosis, we performed laser-induced choroidal neovascularization (CNV) lesions in C57Bl6 wild type and PlGF-DE-Kin mice. Subretinal fibrosis, observed by Collagen I staining after 21 and 35 days after laser damage, is severely impaired in PlGF-DE mice compared to C57Bl6 wild type mice. This reduction is accompanied by alteration of the expression of EndEMT markers, as evaluated by qRT-PCR. Indeed, we observed a reduction of endothelial transcript Cd31 mRNA and increased of mesenchymal markers Fibronectin, Vimentin, Acta2 (Smooth muscle alpha-actin) and Transgelin mRNAs. Therefore, we have harvested total RNA from RPE/choroid tissue and we have performed RNAseq experiments on biological triplicates. In addition, we set up the protocol to isolate mouse choroidal endothelial cells (mCECs) and we are harvesting mCECs from C57Bl6 wild type and PlGF-DE-Kin mice in order to generate an in vitro system to study EndEMT.
To further explore the role of Cripto in regulating EndMT, we have performed Single Cell RNA-Seq analysis on mononuclear cell suspensions isolated from adult hindlimb muscles of control and myeloid-specific Cripto LOF mice at different time points after-injury. Bioinformatic analysis is currently in progress.
To further understand whether the reduction of the cMoP population (osteoclast precursors) is due to a cell-autonomous effect or rather results from inefficient support from bone marrow-derived mesenchymal stromal cells (BM-MSCs), we performed RNA-sequencing on cells derived from the bone marrow of Zfp687-/- and Zfp687P937R/P937R mice. Specifically, we collected MSCs from the bone marrow flushed out from long bones (femurs and tibiae) of 8-week-old mice. BM-MSCs were then cultured in a growth medium designed to promote their enrichment while maintaining their tri-lineage differentiation potential. The MSCs were then cultured for seven days, followed by RNA extraction after removing hematopoietic contaminants using flow cytometry (CD45- and CD11b- cells). Bioinformatic analysis is currently in progress.
Lev R, Bar-Am O, Saar G, Guardiola O, Minchiotti G, Peled E, Seliktar D. Cell Death Dis. 2024 Jul 2;15(7):470. doi: 10.1038/s41419-024-06645-2
Tarallo V, Magliacane Trotta S, Panico S, D'Orsi L, Mercadante G, Cicatiello V, De Falco S. Invest Ophthalmol Vis Sci. 65(8):12. (2024)
Pisapia L, Mercadante V, Andolfi G, Minchiotti G, Guardiola O. Protocol for characterizing non-genetic heterogeneity and expression dynamics of surface proteins in mouse muscle stem cells using flow cytometry. STAR Protoc. 2024 Sep 20;5(3):103216. doi: 10.1016/j.xpro.2024.103216. Epub 2024 Jul 27.
Rodríguez C, Timóteo-Ferreira F, Minchiotti G, Brunelli S, Guardiola O. Cellular interactions and microenvironment dynamics in skeletal muscle regeneration and disease. Front Cell Dev Biol. 2024 May 22;12:1385399. doi: 10.3389/fcell.2024.1385399.