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.
To further investigate the role of ZNF687 in modulating fate-commitment and the differentiation potential of hematopoietic stem and progenitors cells (HSCPs – Ter119-B220-CD117+), we performed single cell RNA sequencing (scRNA-seq) on sorted cells isolated from bone marrow (BM) of Zfp687-/- and wild-type (WT) mice. Interestingly, preliminary analysis revealed a marked reduction in the number of cellular clusters in Zfp687-/- samples, with 18 distinct cellular clusters identified in WT mice compared to only 13 clusters in Zfp687-/- mice. The reduction suggests a loss of cellular heterogeneity within the hematopoietic lineage upon Zfp687 deficiency. Further investigations are currently underway to identify which specific cell clusters are lost in the Zfp687-/- BMs and to elucidate the deregulated molecular mechanisms underlying this reduced heterogeneity.
We have proceeded with scRNA-seq analysis on mononuclear cell suspensions isolated from adult skelethal muscles of control (LyZCre) and myeloid-specific Cripto Loss-of-Function (CriptoKO) mice at 3 and 5 days post-injury (dpi). Bioinformatic analysis, including UMAP dimensional reduction and clustering, facilitated the annotation of major cell populations within the injured muscle tissue. These populations include macrophages, fibro-adipogenic progenitors (FAPs), endothelial cells, muscle stem cells (MuSCs), fibroblasts, smooth muscle and mural cells (SMMCs), glial cells, natural killer (NK) cells, dendritic cells, T cells, B cells, granulocytes, and neutrophils. An additional cell population with lower gene and read counts was also identified and is currently under investigation for its potential biological relevance. Analysis of cell cycle related genes indicated that a significant proportion of both MuSCs and FAPs were captured in the G1 phase across all samples, potentially reflecting the isolation of quiescent populations. Comparative analysis of cell type distribution across conditions and time points revealed a significant increase in the proportion of fibroblasts in wt mice compared to myeloid-specific Cripto LOF mice at 5 dpi. Differential gene expression analysis of macrophages from myeloid-specific Cripto LOF mice revealed a significant upregulation of genes potentially involved in stress response (Hspa1a, Hspa1b), cell-cell and cell-matrix interactions (Vill1, Ctnnd2, Negr1, Marco), growth factor signaling (Ptn), and immune modulation (Ido2), suggesting a complex adaptation of macrophage function in the absence of myeloid Cripto. Conversely, Ighg2c was among the most downregulated genes, along with Mlh1, Vrk2, Mmp19, and Gab1, indicating potential alterations in fundamental cellular processes within macrophages lacking Cripto. Bioinformatic analysis is ongoing to further explore these preliminary findings and gain a deeper understanding of how myeloid Cripto regulates the cellular landscape and specific cell populations during skeletal muscle regeneration.
To investigate subretinal fibrosis, we established a protocol to isolate mouse choroidal endothelial cells (mCECs) from C57Bl6 wild-type (WT) and PlGF-DE-Kin mice, generating an in vitro system to study Endothelial-to-Mesenchymal Transition (EndEMT). After isolation and culture, we induced EndEMT by treating WT mCECs with mouse TGF-β2 (10 ng/mL) for five days. Following treatment, the cells underwent a complete phenotypic shift, adopting a spindle-like morphology consistent with mesenchymal characteristics. Western blot analysis confirmed these changes, revealing increased protein expression of the mesenchymal markers ACTA2, COL1 and Fibronectin and decreased expression of the endothelial marker VE-Cadherin in TGF-β2-treated mCECs compared to vehicle-treated controls. qRT-PCR further supported these findings, showing a significant upregulation of the mesenchymal marker Acta1, Col1, Tagln, Fn and Vim. Currently, we are isolating and culturing mCECs from PlGF-DE-Kin mice (mCECs-DE) to assess whether this transition is altered in cells expressing the dysfunctional PlGF variant.