Combining Nanomaterials and Developmental Pathways to Design New Treatments for Cardiac Regeneration: The Pulsing Heart of Advanced Therapies

Abstract

The research for heart therapies is challenged by the limited intrinsic regenerative capacity of the adult heart. Moreover, it has been hampered by the poor results obtained by tissue engineering and regenerative medicine attempts at generating functional beating constructs able to integrate with the host tissue. For this reason, organ transplantation remains the elective treatment for end-stage heart failure, while novel strategies aiming to promote cardiac regeneration or repair lag behind. The recent discovery that adult cardiomyocytes can be ectopically induced to enter the cell cycle and proliferate by a combination of microRNAs and cardioprotective drugs, like anti-oxidant, anti-inflammatory, anti-coagulants and anti-platelets agents, fueled the quest for new strategies suited to foster cardiac repair. While proposing a revolutionary approach for heart regeneration, these studies raised serious issues regarding the efficient controlled delivery of the therapeutic cargo, as well as its timely removal or metabolic inactivation from the site of action. Especially, there is need for innovative treatment because of evidence of severe side effects caused by pleiotropic drugs. Biocompatible nanoparticles possess unique physico-chemical properties that have been extensively exploited for overcoming the limitations of standard medical therapies. Researchers have put great efforts into the optimization of the nanoparticles synthesis and functionalization, to control their interactions with the biological milieu and use as a viable alternative to traditional approaches. Nanoparticles can be used for diagnosis and deliver therapies in a personalized and targeted fashion. Regarding the treatment of cardiovascular diseases, nanoparticles-based strategies have provided very promising outcomes, in preclinical studies, during the last years. Efficient encapsulation of a large variety of cargos, specific release at the desired site and improvement of cardiac function are some of the main achievements reached so far by nanoparticle-based treatments in animal models. This work offers an overview on the recent nanomedical applications for cardiac regeneration and highlights how the versatility of nanomaterials can be combined with the newest molecular biology discoveries to advance cardiac regeneration therapies.

Keywords: Hippo pathway; YAP; cardiac regeneration; cardiomyopathy; nanoparticles; targeted delivery.

FIGURE 1

HCM and DCM as the most common cardiomyopathies. (A) Schematic representations of the anatomy of normal (left), hypertrophic (hypertrophic cardiomyopathy, HCM, middle) and dilated (dilated cardiomyopathy, DCM, right) heart. HCM is characterized by left ventricle thickening, while DCM is defined by the dilation of the left ventricle. AO, aorta; SVC, superior vena cava; MPA, main pulmonary artery; RA, right atrium; RV, right ventricle; TV, tricuspid valve; PV, pulmonary valve; LA, left atrium; LV, left ventricle; MV, mitral valve; AOV, aortic valve; IVC, inferior vena cava. (B) Hematoxylin-eosin (H&E, top) staining in longitudinal and transversal (inset) sections of the cardiac muscle. Normal heart shows the organized and parallel alignment of cardiomyocytes (dashed black line) with preserved cell body and sarcomeric structures. Disorganized cellular structures are highlighted in HCM and DCM sections. Nuclei appear in violet, cytoplasm in pink. Masson’s trichrome (MT, bottom) staining in longitudinal and transversal (inset) sections of cardiac muscle. The homogeneity, the continuity and organization of the healthy tissue (pink) is perturbed by the accumulation of fibrotic non-contractile tissue (blue) in HCM and DCM hearts. Missing scale bars were not provided in the original article. H&E healthy: Reprinted and adapted from Marian and Braunwald (2017) under the terms of the Creative Commons Attribution License. MT healthy: Reprinted and adapted from Wang et al. (2014) under the terms of the Creative Commons Attribution License. H&E HCM: Reprinted and adapted from Jain et al. (2017) under the terms of the Creative Commons Attribution License. MT HCM: Reprinted and adapted from: Guo et al. (2017) under the terms of the Creative Commons Attribution License. H&E DCM: Jain et al. (2017) under the terms of the Creative Commons Attribution License. MT DCM: Reprinted and adapted from: Mitrut et al. (2018) under the terms of the Creative Commons Attribution License.

FIGURE 2

The role of Hippo pathway in heart development and homeostasis. (A) When Hippo pathway is on, the mammalian MST1/2 (STE20-like protein kinase 1/2) and SAV1 (protein salvador homolog 1) complex activates LATS1/2 (large tumor suppressor homolog 1/2) that phosphorylates, in association with MOB1 (MOB kinase activator 1), YAP (Yes-associated protein 1) and TAZ (WW domain-containing transcription regulator protein 1), thus promoting their degradation. Conversely, when Hippo signaling is off, YAP and TAZ shuttle into the nucleus where they bind TEADs (TEA domain transcription factor family members) and regulate the transcription of genes involved in cell proliferation, survival and migration (Henry et al., 2001). By regulating the activity of YAP and consequently the proliferation of prenatal cardiomyocytes, Hippo pathway regulates heart size during development. YAP inhibitory kinase TAOK1 (TAO kinase 1) and E3 ubiquitin ligase β-TrCP (β-transducin repeats-containing protein) are involved in YAP degradation. Their silencing has been reported to increase YAP activity in cardiomyocytes (Torrini et al., 2019). (B) Increased YAP activity has been associated with several pro-regenerative effects in developed heart to prevent cardiac diseases or promoting restoration after MI, as indicated in the table (Watt et al., 2015).

FIGURE 3

Therapy at the nanoscale. (A) Representation of the nanosize, scaling down from the organ size (swine heart) to single cells and intracellular organelles, the latter being the group in which NPs are included. (B) Illustrative scheme of the different modalities of drug delivery. NPs as drug delivery systems have plenty of advantages if compared to systemic drug administration, such as targeted release and dosage reduction, thanks to the combination of several functional blocks within the same tool.

FIGURE 4

NPs for cardiac diseases. (A) The administration routes currently evaluated for delivery therapy at heart include inhalation (in.), intra-cardiac injection (i.c.) and intra-venous injection (i.v.). (B) Table showing advantages and disadvantages of the different administration routes. (C) The infarcted area typically shows loss of vessels and various degrees of fibrotic scar formation with limited contractility and functionality that dramatically compromise heart functions. NPs can be conceived to help restoring vascularization by promoting angiogenesis (1), locally transdifferentiating fibroblasts into CMs, resolving the fibrotic scar (2), and promoting the anti-inflammatory polarization of immune system cells, like macrophages (Mφ) and monocytes (Mo). The main text provides detailed discussion for each of the points here briefly described.

FIGURE 5

Polymeric NPs for cardiac repair. (A) Schematic representation of polymeric NPs with overall composition and structure. The use of amphiphilic building block allows the formation of micelle-like structures in aqueous media. (B) Advantages and disadvantages associated with the use of polymeric nanoparticles in term of synthesis, long-term stability and clinical translation of the material. (C) Top. Polymeric nanoparticles carrying miRNAs (miNPs) are significantly less toxic for human embryonic stem cell-derived cardiomyocytes when compared to lipofectamine (lipo). Alpha-actinin (green) stains the contractile structure and Ki67 (red) accounts for the proliferation capacity. Nuclei are counterstained by DAPI (blue). Bottom. Picro Sirius Red ex vivo staining of heart transversal tissue sections showing a clear reduction of the scar size and fibrosis in left ventricle for the heart treated with miNPs (right) in comparison to the same particles carrying a scramble miRNA (left). (D) Left. miRNA-21 NPs myocardial infarction treatment causes increased levels of anti-inflammatory cytokine TGF-β both at infarction border zone (top) and in remote myocardium (bottom) when compared to saline-treated or control. Center. miRNA-21 NPs induce neovascularization in infarcted mouse myocardium, as shown by CD31 + blood vessels in cross-sections of the heart (dotted black box) and quantified in the graph by comparing it with saline and control NPs-treated groups *p < 0.05. Right. Histological analysis shows the presence of macrophages in infarcted heart treated with miRNA-21 NPs, as shown by CD11b-positive cells positive cells (black arrow), but no macrophage accumulation as compared to saline and control NPs as shown in the graph. Reprinted from: (C) (Yang et al., 2019) with permission from American Chemical Society; (D) (Bejerano et al., 2018) with permission from American Chemical Society.

FIGURE 6

Liposomes for active targeting of infarcted heart. (A) Schematic representation of liposomes with overall composition and structure. (B) Advantages and disadvantages associated with the use of liposomes. (C) Top. In vivo bioluminescence images of infarcted mouse hearts injected either with AT1 or scrambled (S) liposomes and analyzed at the indicated timepoints indicate AT1-liposomes preferential accumulation in the tissue (orange/red signal). The graph shows the quantification of the fluorescence due to AT1- and scramble-liposomes accumulation at the given timepoints. (D) Top left. Dil-labeled-Platelet-like proteoliposomes (PLPs) selectively interact with RAW264.7 monocytic cell line and macrophages (Mϕ) but not with SVECs endothelial cells, as indicated by the red signal. Scale bar 10 μm. Dil = 1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindocarbocyanine Perchlorate. Bottom left. Intravenously injected PLPs accumulate in the heart more efficiently than liposomes 72 h post-infarction *p < 0.05, **p < 0.01. Right. The persistence of Dil-labeled PLPs in the infarction area in a mouse model of ischemia/reperfusion (I/R + PLPs, red square) as compared to Dil-labeled liposomes (I/R + Liposomes, white square) and sham control. Troponin I (TnI, green), stains the heart muscle, Dil (red) injected liposomes, cell nuclei are counterstained with DAPI (blue), Scale bar were not reported in the original article. Reprinted and adapted from: (C) (Dvir et al., 2011) with permission from American Chemical Society; (D) (Cheng et al., 2016) with permission from Wiley.

FIGURE 7

Inorganic NPs for cardiac regeneration. (A) Schematic representation of inorganic NPs used for cardiac regeneration. (B) Table listing the advantages and disadvantages associated with the use of inorganic nanoparticles. (C) Representative hematoxylin/eosin (cell nuclei and cytoplasm) and Prussian blue (iron oxide nanoparticles) images of infarcted myocardium (MI) injected with endothelial progenitor cells loaded with iron oxide nanoparticles (Fe-EPCs) and exposed (+M) or not to external magnetic field. The graph quantifies the retention of EPCs and Fe-EPCs in the presence or not of magnetic field *p < 0.05. (D) Representative Masson’s trichrome staining of infarcted myocardium (MI) injected with endothelial progenitor cells loaded (Fe-EPCs) or not (EPCs) with iron oxide nanoparticles and exposed (+M) or not to external magnetic field. Blue color and black arrow identify the fibrotic area. The graph quantifies the infarction area as obtained by image analysis. *p < 0.05 versus the control Sham operated group (not shown), ^#p < 0.05 versus the other group shown in the graph. Reprinted and adapted from: (C,D) (Zhang et al., 2019) with permission of Wiley.