ex-vivo-myocardial-infarction-model-crystalloid-perfused-mouse

<em>Ex Vivo</em> Myocardial Infarction Model in the Crystalloid-Perfused Mouse Heart

Experimental myocardial infarction (MI) is commonly modeled in animals through transient occlusion of the left anterior descending (LAD) coronary artery ...

Affiliated Danyang Hospital of Nantong University

The pericardial adipose derived stromal cells (pADSC) own a developmental origin from the "second heart field" and thus favor myogenic differentiation. The present experiments extended our previous observation by defining a subset of pADSC marked with the expression of flk-1, a type II receptor for VEGF to efficiently enhance cardiac repair.

Reconstitute the damaged heart via the dual reparative roles of pericardial adipose-derived flk-1+ stem cells.

Injury may induce a sequential activation of intrinsic reparative activity that supports the maintenance of tissue homeostasis.

Injury-induced fetal reprogramming imparts multipotency and reparative properties to pericardial adipose stem cells.

Acute myocardial infarction (AMI) evokes a temporally coordinated immune response, in which monocytes are critically involved in the clearance of cell debris; however, excessive inflammation induced by the classical sub-population of monocytes frequently limits the endogenous reparative process. In the present study, the potential of the anti-inflammatory adipokine complement C1q tumor necrosis factor (TNF)-related protein-3 (CTRP3) to induce intermediate switch of monocytes to an anti-inflammatory phenotype was explored. Circulating monocytes were isolated from patients with AMI at various time-points (3-5 h, 3 days and 7 days) and categorized by flow cytometry/immunostaining into three sub-divisions based on the expression of CD14 and CD16 epitopes: Classical (CD14/CD16), non-classical (CD14/CD16) and intermediate populations (CD14/CD16). The phagocytic activity was evaluated by the ingestion of FITC-Zymosan and F-nanoemulsion and the migratory activity using Thin Cert™ Transwell assay. Monocytes were cultured using autologous serum in the presence of CTRP3 (1 µg/ml) for 24 h and the expression of interleukin 6 (IL-6) and TNF-α was quantified by reverse-transcription quantitative PCR. In addition, SB203580, a p38 mitogen-activated protein kinase (MAPK)/ERK inhibitor, was used to examine the downstream pathways of CTRP3. AMI evoked a transient increase in monocyte counts of the classical subset after onset of the ischemic insult, while the non-classical and intermediate subsets persistently expanded (P<0.01). The monocytes from patients at 3 days after AMI displayed enhanced phagocytic and migratory activities in comparison with those from healthy volunteers (P<0.01). Of note, addition of CTRP3 induced an intermediate switch of monocyte subsets and antagonized the enhanced expression of cytokines, particularly IL-6, in monocytes stressed by lipopolysaccharides, likely by blunting the ERK1/2 and P38 MAPK signaling pathway. In conclusion, the present study demonstrated a dynamic fluctuation of monocyte subsets and enhanced phagocytic and migratory activities in patients with AMI. Furthermore, the 'proof-of-concept' evidence pinpoints CTRP3 as an alternative candidate to modulate the 'uncontrolled' inflammatory response and thus to augment cardiac reparative processes in patients with AMI.

CTRP3 induces an intermediate switch of CD14CD16 monocyte subset with anti-inflammatory phenotype.

Cellular dedifferentiation is a regenerative prerequisite that warrants cell cycle reentry and appropriate mitotic division during de novo formation of cardiomyocytes. In the light of our previous finding that expression of injury-responsive element, Wilms Tumor factor 1 (WT1), in pericardial adipose stromal cells (ADSC) conferred a compelling reparative activity with concomitant IL-6 upregulation, we then aim to unravel the mechanistic network that governs the process of regenerative dedifferentiation after ADSC-based therapy.

IL-6 coaxes cellular dedifferentiation as a pro-regenerative intermediate that contributes to pericardial ADSC-induced cardiac repair.

Tissue damage often induces local inflammation that in turn dictates a series of subsequential responses, such as stem cell activation and growth, to maintain tissue homeostasis. The aim of the study is to testify the possibility of using inflammation-trained stem cells as optimal donor cells to augment the efficacy of cell therapy. The pericardial stem/stromal cells derived from the animals after myocardial infarction (MI-pSC) showed an enhanced myogenic potential and augmented reparative activity after transplantation in the injured hearts, as compared to the Sham-pSC. Bulk RNA-Seq analysis revealed significant upregulation of a panel of myogenic and trophic genes in the MI-pSC and, notably, Sfrp1 as an important anti-apoptotic factor induced robustly in the MI-pSC. Injection of the MI-pSC yielded measurable numbers of surviving cardiomyocytes (Tunel and Casp-3 negative) within the infarct area, but the effects were significantly diminished by siRNA-based silence of Sfrp1 gene in the pSC. Primed Sham-pSC with pericardial fluid from MI rats mimicked the upregulation of Sfrp1 and enhanced myogenic potential and reparative activity of pSC. Taken together, our results illustrated the inflammation-trained pSC favor a reparative activity through upregulation of Sfrp1 gene that confers anti-apoptotic activity in the injured cardiomyocytes. Therefore, the active form of stem cells may be used as a cardiac protective agent to boost therapeutical potential of stem cells.

Sfrp1 as a Pivotal Paracrine Factor in the Trained Pericardial Stem Cells that Foster Reparative Activity.

Cellular therapy holds immense promise to remuscularize the damaged myocardium but is practically hindered by limited allogeneic sources of cardiac-committed cells that engraft stably in the recipient heart after transplantation. Here, we demonstrate that the pericardial tissue harbors myogenic stem cells (pSCs) that are activated in response to inflammatory signaling after myocardial infarction (MI). The pSCs derived from the MI rats (MI-pSCs) show  and  cardiac commitment characterized by cardiac-specific Tnnt2 expression and formation of rhythmic contraction in culture. Bulk RNA-seq analysis reveals significant upregulation of a panel of genes related to cardiac/myogenic differentiation, paracrine factors, and extracellular matrix in the activated pSCs compared to the control pSCs (Sham-pSCs). Notably, we define MyoD as a key factor that governs the process of cardiac commitment, as siRNA-mediated  gene silencing results in a significant reduction of myogenic potential. Injection of the cardiac-committed cells into the infarcted rat heart leads to long-term survival and stable engraftment in the recipient myocardium. Therefore, these findings point to pericardial myogenic progenitors as an attractive candidate for cardiac cell-based therapy to remuscularize the damaged myocardium.

Cardiac commitment driven by MyoD expression in pericardial stem cells.

Histological analysis with 2,3,5-triphenyltetrazolium chloride (TTC) staining is the most frequently used tool to detect myocardial ischemia/reperfusion injury. However, its practicality is often challenged by poor image quality in gross histology, leading to an equivocal infarct-boundary delineation and potentially compromised measurement accuracy. Here, we introduce several crucial refinements in staining protocol and sample processing, which enable TTC images to be analyzed with light microscopy. The refined protocol involves a two-step TTC staining process (perfusion and immersion) and subsequent Zamboni fixation to differentiate myocardial viability and necrosis, and use of Coomassie brilliant blue to label area-at-risk. After the duo-staining steps were completed, the heart sample was embedded and sliced transversally by a cryostat into a series of thin sections (50 µm) for microscopic analysis. The refined TTC (redTTC) assay yielded remarkably high-quality images with striking color intensity and sharply defined boundaries, permitting unambiguous and reliable delineation of the infarct and area-at-risk. In the same animals, the redTTC assay showed good agreement with the in-vivo gold standard measurements (LGE and MEMRI). Meanwhile, redTTC imaging allows tracking of viable cardiomyocytes at cellular resolution, and with this enhanced capability, we convincingly demonstrated the pro-survival action of stem cells based-therapy. Therefore, the redTTC assay represents a significant technical advance that permits precise detection of the true extent of cardiac injury and cardiomyocyte viability. This approach is cost-effective and may be adapted for use in diverse applications, making it highly appealing to many laboratories performing ischemia/reperfusion injury experiments.

A refined TTC assay precisely detects cardiac injury and cellular viability in the infarcted mouse heart.

With the growing need for reliable and precise detection of cell viability in spatial biology, we introduce an antibody-based staining of cardiac troponin I (cTnI) as a simple yet valuable tool for delineating cardiomyocyte viability in the early stages of myocardial infarction (MI).

Article	Year
Reconstitute the damaged heart via the dual reparative roles of pericardial adipose-derived flk-1+ stem cells. International journal of cardiology\| PubMed ID: 26407047	2016
Injury-induced fetal reprogramming imparts multipotency and reparative properties to pericardial adipose stem cells. Stem cell research & therapy\| PubMed ID: 30103817	2018
CTRP3 induces an intermediate switch of CD14CD16 monocyte subset with anti-inflammatory phenotype. Experimental and therapeutic medicine\| PubMed ID: 32104290	2020
IL-6 coaxes cellular dedifferentiation as a pro-regenerative intermediate that contributes to pericardial ADSC-induced cardiac repair. Stem cell research & therapy\| PubMed ID: 35101092	2022
Sfrp1 as a Pivotal Paracrine Factor in the Trained Pericardial Stem Cells that Foster Reparative Activity. Stem cells translational medicine\| PubMed ID: 37936560	2024
Cardiac commitment driven by MyoD expression in pericardial stem cells. Frontiers in cell and developmental biology\| PubMed ID: 38601082	2024
A refined TTC assay precisely detects cardiac injury and cellular viability in the infarcted mouse heart. Scientific reports\| PubMed ID: 39448689	2024
An immunostaining-based approach for assessing myocardial viability in the infarcted mouse hearts. Frontiers in cardiovascular medicine\| PubMed ID: 40529549	2025

Xueqing Liu

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