By adulthood, the heart is no longer able to replenish injured or diseased cells but may be possible to reverse this damage and restore heart function


By adulthood, the heart is no longer able to replenish injured or diseased cells. As a result, heart disease or an event like a heart attack can be disastrous, leading to massive cell death and permanent declines in function.

A new study by scientists at the Lewis Katz School of Medicine at Temple University (LKSOM), however, shows that it may be possible to reverse this damage and restore heart function, even after a severe heart attack.

The study, published June 21 in the print edition of the journal Circulation Research, is the first to show that a very small RNA molecule known as miR-294, when introduced into heart cells, can reactivate heart cell proliferation and improve heart function in mice that have suffered the equivalent of a heart attack in humans.

“In previous work, we discovered that miR-294 actively regulates the cell cycle in the developing heart,” said Mohsin Khan, Ph.D., Assistant Professor of Physiology at the Center for Metabolic Disease Research at LKSOM. “But shortly after birth miR-294 is no longer expressed.”

Dr. Khan and colleague Raj Kishore, Ph.D., Professor of Pharmacology and Medicine and Director of the Stem Cell Therapy Program in the Center for Translational Medicine at LKSOM, both senior investigators on the new study, wondered whether miR-294 could serve as a sort of fountain of youth for heart cells.

“The heart is very proliferative when miR-294 is expressed in early life,” Dr. Kishore explained.

“We wanted to see if reintroducing it into adult heart cells would turn them back to an embryonic-like state, allowing them to make new heart cells.”

The researchers tested their idea in mice that had myocardial infarction (heart attack). Mice were treated with miR-294 continuously for two weeks after sustaining myocardial injury.

Two months following treatment, the researchers observed noticeable improvements in heart function and a decrease in the area of damaged tissue.

Examination of treated heart cells revealed evidence of cell cycle reentry, indicating that the cells had been reactivated, regaining the ability to produce new cells.

“The miR-294 treatment reawakened an embryonic signaling program in the adult heart cells,” said Dr. Khan.

“Because of this, the old heart cells were no longer quite like adult cells, but neither were they fully embryonic. In this in-between state, however, they had the ability to make new cells.”

The researchers were able to control miR-294 expression, turning it on or off and thereby dictating the amount of proliferative activity in the heart.

Drs. Khan and Kishore plan next to replicate the study in a large animal model. They also want to gain a deeper understanding of what miR-294 is doing in the heart.

“There is evidence that it does more than control the cell cycle,” Dr. Khan said. “If it has multiple targets, we need to find them.”

In the last few years a variety of stem cells have been scrutinized for cardiac repair, including peripheral blood-derived progenitor cells, BM- and adipose-derived MSC, fetal and perinatal stem and progenitor cells, embryonic and iPSC (as extensively reviewed in Abdelwahid et al., 2016).

Given the promising cardioprotective and pro-angiogenic potential of stem cell-EV/Ex, many efforts have recently been focused on identifying the ideal cell source for scaling up the production of EV and Ex as advanced medicinal product for ischemic-related diseases.

Cardiovascular disease patients need prompt therapeutic intervention and it would be ideal to have access to regenerative “off-the-shelf” products for simple administration; hence, stem cells might be used as a “drug store” to produce a highly efficient EV/Ex formulation to provide enhancement of cardiac repair (Figure ​(Figure1).1).

In this scenario, isolation feasibility and elevated self-renewal represents key aspects of the optimal stem cell source to be exploited for future paracrine therapy.

Human adult progenitor cells, including MSC, can be isolated from a variety of post-natal tissues and obtained from discarded samples as clinical waste or leftover material during ordinary surgical or screening procedures (blood sampling, liposuction, BM transplantation, etc.).

However, they can be affected by low yield and limited self-renewal potential, as they are often influenced by donor age (van Vliet et al., 2010).

On the contrary, iPSC may overcome classical drawbacks of adult MSC by offering, in principle, unlimited production of stem and progenitor cells in vitro.

Indeed, when comparing cardiac reparative effects of murine iPSC against their secreted EV, the latter showed superior paracrine potential in a preclinical model of I/R injury in mouse, thus representing an appealing therapeutic option by offering the benefits of iPSC therapy, but without the risk of tumorigenicity (Adamiak et al., 2017).

Yet, iPSC technology can be challenging, costly and time consuming.

The more recent characterization of fetal (with non-embryonic origin) and perinatal progenitor cells, have broadened the options.

Fetal stem cells isolated either from AF (De Coppi et al., 2007) or villi (Poloni et al., 2008) as easily collected from leftover sample obtained during routine prenatal screening; perinatal stem cells can be isolated at birth from discarded extra-embryonic annexes, such as UC, including the WJ (Wang et al., 2004Tauro et al., 2012), and placenta membranes (Magatti et al., 2016), thus representing an easily accessible source progenitors available in large amount and free from any ethical concern.

Fetal and perinatal stem cells can offer specific advantages over adult MSC, since they are endowed with outstanding self-renewal and possibly higher paracrine potential than the adult ones, being developmentally more immature.

Indeed, human UC-MSC-EV systemically injected into a preclinical rat model of MI sustained cardiac systolic function after 4 weeks, while offsetting fibrosis and cells apoptosis (Zhao et al., 2015).

Specific interest has also been recently dedicated to the paracrine potential of human amniotic fluid-derived stem cell-EV, as described by few independent studies mediating therapeutic pro-survival, angiogenic, and anti-inflammatory effects (Balbi et al., 2017Mellows et al., 2017Sedrakyan et al., 2017Beretti et al., 2018).

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Schematic representation of the most relevant effects of extracellular vesicle-mediated intercellular communication within the injured myocardium as providing: (A) cardio-protection and cardiac repair by exogenous stem cell-EV/Ex; (B) paracrine modulation of the cardiac microenvironment by EV/Ex derived from CPC either activated in situ (i) or previously isolated and expanded in vitro (ii); and (C) fine tuning of adult resident cardiomyocyte and cardiac stromal cell behavior by their mutual EV/Ex modulatory exchange during adaptive response to stress. ATMP, advanced therapy medicinal product.

Stromal cell function and secretory potential can be critically determined by the microenvironment scenario.

Notably, fine tuning of the microenvironment in vitro can significantly and quantitatively influence the cell secretome (Leuning et al., 2018).

Therefore, in vitro cell-preconditioning can be adjusted in order to select the ideal cell culture conditions to prime stem cell-EV with optimal cardioprotective and pro-angiogenic potential for therapeutic relevance. For instance, short term hypoxic stimulation has been increasingly used to enrich MSC-EV and Ex with trophic paracrine factors.

Delivery of these vesicles resulted in promotion of neovascularization and cardiac repair in preclinical rodent models of MI.

The putative mechanisms of action have been reported to involve increased expression of nSMase2, which is critical for exosome biogenesis and EV-mediated transfer of miR-210 (Bian et al., 2014Gonzalez-King et al., 2017Zhu et al., 2017).

Likewise, transgenic overexpression of specific cardio-active factors within stem cells has been employed to enhance the healing capacity of their secreted EV; overexpression of HIF-1α in human dental pulp-MSC led to secretion of enhanced Jagged 1-loaded exosomes that triggered the angiogenic differentiation of endothelial cells (Gonzalez-King et al., 2017).

Moreover, Li et al. (2010) previously showed that rat (bone marrow) BM-MSC genetically modified to significantly increase GATA-4 expression, are endowed with noteworthy paracrine pro-survival and angiogenic potential in the ischemic environment.

They then exploited the same working strategy to boost the cardioprotective paracrine effect of MSC-Ex in a preclinical rodent model of I/R injury; exosomal reprogramming of resident cardiomyocytes via the delivery of miR-19a targeting PTEN and BIM protein expression resulted in sustained activation of Akt and ERK signaling pathway, with significant recovery of cardiac function and decreased infarct size (Yu et al., 2015).

Stem cell-EV are being increasingly considered as biological enhancers of heart repair mechanisms (Madonna et al., 2016), yet little is known about their cardiac regenerative potential to trigger functional activation of endogenous CPC and responsive resident cardiomyocyte proliferation.

Currently only a few studies have provided evidences of such critical effects.

Mouse ESC-Ex have been demonstrated to prompt rodent resident c-KIT+ CPC survival, proliferation, and cardiac commitment several weeks after in vivo administration, with restitution of de novo cardiomyocytes in the ischemic heart, possibly via specific miR-294 transfer (Khan et al., 2015).

Similarly, human iPSC-secreted shedding MV exerted in vitro proliferative and protective effects on human cardiac MSC possibly via direct transfer of miR-92b and elicited their cardiac and endothelial differentiation potential (Bobis-Wozowicz et al., 2015).

Moreover, rat BM-MSC-Ex showed to prime c-KIT+ rat neonatal CPC by reprogramming their miRNA landscape, thus targeting a broad range of biological functions, from positive regulation of cell cycle, up to cell differentiation and response to hypoxia; such MSC-Ex preconditioning strategy also boosted CPC angiogenic ability and increased their in vivo functional potency after transplantation into the rat ischemic myocardium (Zhang et al., 2016).

While such data is surely encouraging and very promising, further comprehensive studies are required to specifically assess stem cell-EV potential to sustain functional restoration of myocardial renewal via cardiomyocyte proliferation as well.

While standard cell therapy has been almost completely dismissed, high expectations have now been put into the therapeutic efficacy of stem cell-EV as paracrine facilitators of cardiac repair and regeneration.

Indeed, they may represent an appealing alternative cell-free curative modality that could be clinically and translationally effective, safer, and cheaper. However, such a novel approach is still in its infancy as it requires extensive testing to validate EV safety and functional efficacy in the long term.

More information: Austin Borden et al, Transient Introduction of miR-294 in the Heart Promotes Cardiomyocyte Cell Cycle Reentry After Injury, Circulation Research (2019). DOI: 10.1161/CIRCRESAHA.118.314223

Journal information: Circulation Research
Provided by Temple University


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