Small Extracellular Vesicles From Infarcted and Failing Heart Accelerate Tumor Growth.

BACKGROUND: Myocardial infarction (MI) and heart failure are associated with an increased incidence of cancer. However, the mechanism is complex and unclear. Here, we aimed to test our hypothesis that cardiac small extracellular vesicles (sEVs), particularly cardiac mesenchymal stromal cell-derived sEVs (cMSC-sEVs), contribute to the link between post-MI left ventricular dysfunction (LVD) and cancer. METHODS: We purified and characterized sEVs from post-MI hearts and cultured cMSCs. Then, we analyzed cMSC-EV cargo and proneoplastic effects on several lines of cancer cells, macrophages, and endothelial cells. Next, we modeled heterotopic and orthotopic lung and breast cancer tumors in mice with post-MI LVD. We transferred cMSC-sEVs to assess sEV biodistribution and its effect on tumor growth. Finally, we tested the effects of sEV depletion and spironolactone treatment on cMSC-EV release and tumor growth. RESULTS: Post-MI hearts, particularly cMSCs, produced more sEVs with proneoplastic cargo than nonfailing hearts did. Proteomic analysis revealed unique protein profiles and higher quantities of tumor-promoting cytokines, proteins, and microRNAs in cMSC-sEVs from post-MI hearts. The proneoplastic effects of cMSC-sEVs varied with different types of cancer, with lung and colon cancers being more affected than melanoma and breast cancer cell lines. Post-MI cMSC-sEVs also activated resting macrophages into proangiogenic and protumorigenic states in vitro. At 28-day follow-up, mice with post-MI LVD developed larger heterotopic and orthotopic lung tumors than did sham-MI mice. Adoptive transfer of cMSC-sEVs from post-MI hearts accelerated the growth of heterotopic and orthotopic lung tumors, and biodistribution analysis revealed accumulating cMSC-sEVs in tumor cells along with accelerated tumor cell proliferation. sEV depletion reduced the tumor-promoting effects of MI, and adoptive transfer of cMSC-sEVs from post-MI hearts partially restored these effects. Finally, spironolactone treatment reduced the number of cMSC-sEVs and suppressed tumor growth during post-MI LVD. CONCLUSIONS: Cardiac sEVs, specifically cMSC-sEVs from post-MI hearts, carry multiple protumorigenic factors. Uptake of cMSC-sEVs by cancer cells accelerates tumor growth. Treatment with spironolactone significantly reduces accelerated tumor growth after MI. Our results provide new insight into the mechanism connecting post-MI LVD to cancer and propose a translational option to mitigate this deadly association.

CRISPR-Cas9 editing of TLR4 to improve the outcome of cardiac cell therapy.

Inflammation and fibrosis limit the reparative properties of human mesenchymal stromal cells (hMSCs). We hypothesized that disrupting the toll-like receptor 4 (TLR4) gene would switch hMSCs toward a reparative phenotype and improve the outcome of cell therapy for infarct repair. We developed and optimized an improved electroporation protocol for CRISPR-Cas9 gene editing. This protocol achieved a 68% success rate when applied to isolated hMSCs from the heart and epicardial fat of patients with ischemic heart disease. While cell editing lowered TLR4 expression in hMSCs, it did not affect classical markers of hMSCs, proliferation, and migration rate. Protein mass spectrometry analysis revealed that edited cells secreted fewer proteins involved in inflammation. Analysis of biological processes revealed that TLR4 editing reduced processes linked to inflammation and extracellular organization. Furthermore, edited cells expressed less NF-ƙB and secreted lower amounts of extracellular vesicles and pro-inflammatory and pro-fibrotic cytokines than unedited hMSCs. Cell therapy with both edited and unedited hMSCs improved survival, left ventricular remodeling, and cardiac function after myocardial infarction (MI) in mice. Postmortem histologic analysis revealed clusters of edited cells that survived in the scar tissue 28 days after MI. Morphometric analysis showed that implantation of edited cells increased the area of myocardial islands in the scar tissue, reduced the occurrence of transmural scar, increased scar thickness, and decreased expansion index. We show, for the first time, that CRISPR-Cas9-based disruption of the TLR4-gene reduces pro-inflammatory polarization of hMSCs and improves infarct healing and remodeling in mice. Our results provide a new approach to improving the outcomes of cell therapy for cardiovascular diseases.

Osteopontin promotes infarct repair.

Understanding how macrophages promote myocardial repair can help create new therapies for infarct repair. We aimed to determine what mechanisms underlie the reparative properties of macrophages. Cytokine arrays revealed that neonatal cardiac macrophages from the injured neonatal heart secreted high amounts of osteopontin (OPN). In vitro, recombinant OPN stimulated cardiac cell outgrowth, cardiomyocyte (CM) cell-cycle re-entry, and CM migration. In addition, OPN induced nuclear translocation of the cytoplasmatic yes-associated protein 1 (YAP1) and upregulated transcriptional factors and cell-cycle genes. Significantly, by blocking the OPN receptor CD44, we eliminated the effects of OPN on CMs. OPN also activated the proliferation and migration of non-CM cells: endothelial cells and cardiac mesenchymal stromal cells in vitro. Notably, the significant role of OPN in myocardial healing was demonstrated by impaired healing in OPN-deficient neonatal hearts. Finally, in the adult mice, a single injection of OPN into the border of the ischemic zone induced CM cell-cycle re-entry, improved scar formation, local and global cardiac function, and LV remodelling 30 days after MI. In summary, we have shown, for the first time, that recombinant OPN activates cell-cycle re-entry in CMs. In addition, recombinant OPN stimulates multiple cardiac cells and improves scar formation, LV remodelling, and regional and global function after MI. Therefore, we propose OPN as a new cell-free therapy to optimize infarct repair.

PTH-Induced Bone Regeneration and Vascular Modulation Are Both Dependent on Endothelial Signaling.

The use of a bone allograft presents a promising approach for healing nonunion fractures. We have previously reported that parathyroid hormone (PTH) therapy induced allograft integration while modulating angiogenesis at the allograft proximity. Here, we hypothesize that PTH-induced vascular modulation and the osteogenic effect of PTH are both dependent on endothelial PTH receptor-1 (PTHR1) signaling. To evaluate our hypothesis, we used multiple transgenic mouse lines, and their wild-type counterparts as a control. In addition to endothelial-specific PTHR1 knock-out mice, we used mice in which PTHR1 was engineered to be constitutively active in collagen-1α+ osteoblasts, to assess the effect of PTH signaling activation exclusively in osteoprogenitors. To characterize resident cell recruitment and osteogenic activity, mice in which the Luciferase reporter gene is expressed under the Osteocalcin promoter (Oc-Luc) were used. Mice were implanted with calvarial allografts and treated with either PTH or PBS. A micro-computed tomography-based structural analysis indicated that the induction of bone formation by PTH, as observed in wild-type animals, was not maintained when PTHR1 was removed from endothelial cells. Furthermore, the induction of PTH signaling exclusively in osteoblasts resulted in significantly less bone formation compared to systemic PTH treatment, and significantly less osteogenic activity was measured by bioluminescence imaging of the Oc-Luc mice. Deletion of the endothelial PTHR1 significantly decreased the PTH-induced formation of narrow blood vessels, formerly demonstrated in wild-type mice. However, the exclusive activation of PTH signaling in osteoblasts was sufficient to re-establish the observed PTH effect. Collectively, our results show that endothelial PTHR1 signaling plays a key role in PTH-induced osteogenesis and has implications in angiogenesis.

Extracellular Vesicles From Epicardial Fat Facilitate Atrial Fibrillation.

BACKGROUND: The role of epicardial fat (eFat)-derived extracellular vesicles (EVs) in the pathogenesis of atrial fibrillation (AF) has never been studied. We tested the hypothesis that eFat-EVs transmit proinflammatory, profibrotic, and proarrhythmic molecules that induce atrial myopathy and fibrillation. METHODS: We collected eFat specimens from patients with (n=32) and without AF (n=30) during elective heart surgery. eFat samples were grown as organ cultures, and the culture medium was collected every 2 days. We then isolated and purified eFat-EVs from the culture medium, and analyzed the EV number, size, morphology, specific markers, encapsulated cytokines, proteome, and microRNAs. Next, we evaluated the biological effects of unpurified and purified EVs on atrial mesenchymal stromal cells and endothelial cells in vitro. To establish a causal association between eFat-EVs and vulnerability to AF, we modeled AF in vitro using induced pluripotent stem cell-derived cardiomyocytes. RESULTS: Microscopic examination revealed excessive inflammation, fibrosis, and apoptosis in fresh and cultured eFat tissues. Cultured explants from patients with AF secreted more EVs and harbored greater amounts of proinflammatory and profibrotic cytokines, and profibrotic microRNA, as well, than those without AF. The proteomic analysis confirmed the distinctive profile of purified eFat-EVs from patients with AF. In vitro, purified and unpurified eFat-EVs from patients with AF had a greater effect on proliferation and migration of human mesenchymal stromal cells and endothelial cells, compared with eFat-EVs from patients without AF. Last, whereas eFat-EVs from patients with and without AF shortened the action potential duration of induced pluripotent stem cell-derived cardiomyocytes, only eFat-EVs from patients with AF induced sustained reentry (rotor) in induced pluripotent stem cell-derived cardiomyocytes. CONCLUSIONS: We show, for the first time, a distinctive proinflammatory, profibrotic, and proarrhythmic signature of eFat-EVs from patients with AF. Our findings uncover another pathway by which eFat promotes the development of atrial myopathy and fibrillation.

Systemic administration of mesenchymal stem cells combined with parathyroid hormone therapy synergistically regenerates multiple rib fractures.

BACKGROUND: A devastating condition that leads to trauma-related morbidity, multiple rib fractures, remain a serious unmet clinical need. Systemic administration of mesenchymal stem cells (MSCs) has been shown to regenerate various tissues. We hypothesized that parathyroid hormone (PTH) therapy would enhance MSC homing and differentiation, ultimately leading to bone formation that would bridge rib fractures. METHODS: The combination of human MSCs (hMSCs) and a clinically relevant PTH dose was studied using immunosuppressed rats. Segmental defects were created in animals' fifth and sixth ribs. The rats were divided into four groups: a negative control group, in which animals received vehicle alone; the PTH-only group, in which animals received daily subcutaneous injections of 4 µg/kg teriparatide, a pharmaceutical derivative of PTH; the hMSC-only group, in which each animal received five injections of 2 × 106 hMSCs; and the hMSC + PTH group, in which animals received both treatments. Longitudinal in vivo monitoring of bone formation was performed biweekly using micro-computed tomography (µCT), followed by histological analysis. RESULTS: Fluorescently-dyed hMSCs were counted using confocal microscopy imaging of histological samples harvested 8 weeks after surgery. PTH significantly augmented the number of hMSCs that homed to the fracture site. Immunofluorescence of osteogenic markers, osteocalcin and bone sialoprotein, showed that PTH induced cell differentiation in both exogenously administered cells and resident cells. µCT scans revealed a significant increase in bone volume only in the hMSC + PTH group, beginning by the 4th week after surgery. Eight weeks after surgery, 35% of ribs in the hMSC + PTH group had complete bone bridging, whereas there was complete bridging in only 6.25% of ribs (one rib) in the PTH-only group and in none of the ribs in the other groups. Based on the µCT scans, biomechanical analysis using the micro-finite element method demonstrated that the healed ribs were stiffer than intact ribs in torsion, compression, and bending simulations, as expected when examining bone callus composed of woven bone. CONCLUSIONS: Administration of both hMSCs and PTH worked synergistically in rib fracture healing, suggesting this approach may pave the way to treat multiple rib fractures as well as additional fractures in various anatomical sites.

Teriparatide attenuates scarring around murine cranial bone allograft via modulation of angiogenesis.

Nearly all bone fractures in humans can deteriorate into a non-union fracture, often due to formation of fibrotic tissue. Cranial allogeneic bone grafts present a striking example: although seemingly attractive for craniofacial reconstructions, they often fail due to fibrosis at the host-graft junction, which physically prevents the desired bridging of bone between the host and graft and revitalization of the latter. In the present study we show that intermittent treatment with recombinant parathyroid hormone-analogue (teriparatide) modulates neovascularization feeding in the graft surroundings, consequently reducing fibrosis and scar tissue formation and facilitates osteogenesis. Longitudinal inspection of the vascular tree feeding the allograft has revealed that teriparatide induces formation of small-diameter vessels in the 1st week after surgery; by the 2nd week, abundant formation of small-diameter blood vessels was detected in untreated control animals, but far less in teriparatide-treated mice, although in total, more blood capillaries were detected in the animals that were given teriparatide. By that time point we observed expression of the profibrogenic mediator TGF-ß in untreated animals, but negligible expression in the teriparatide-treated mice. To evaluate the formation of scar tissue, we utilized a magnetization transfer contrast MRI protocol to differentiate osteoid tissue from scar tissue, based on the characterization of collagen fibers. Using this method we found that significantly more bone matrix was formed in animals given teriparatide than in control animals. Altogether, our findings show how teriparatide diminishes scarring, ultimately leading to superior bone graft integration.