Biologics and cardiac disease: challenges and opportunities

Biologics are revolutionizing the treatment of chronic diseases, such as cancer and monogenic disorders, by overcoming the limits of classic therapeutic approaches using small molecules. However, the clinical use of biologics is limited for cardiovascular diseases (CVDs) , which are the primary cause of morbidity and mortality worldwide. Here, we review the state-of-the-art use of biologics for cardiac disorders and provide a framework for understanding why they still struggle to enter the field. Some limitations are common and intrinsic to all biological drugs, whereas others depend on the complexity of cardiac disease. In our opinion, delineating these struggles will be valuable in developing and accelerating the approval of a new generation of biologics for CVDs

A new laser device for ultra-rapid and sustainable aerosol sterilization

The current COVID-19 pandemic has highlighted the importance of aerosol-based transmission of human pathogens; this therefore calls for novel medical devices which are able to sterilize contaminated aerosols. Here we describe a new laser device able to sterilize droplets containing either viruses or bacteria. Using engineered viral particles, we determined the 10,600 nm wavelength as the most efficient and exploitable laser source to be manufactured in a commercial device. Given the lack of existing working models to reproduce a human aerosol containing living microbial particles, we developed a new system mimicking human droplet formation and preserving bacterial and viral viability. This evidenced the efficacy of 10,600 nm laser light to kill two aerosol transmitted human pathogens, Legionella pneumophila and SARS-CoV-2. The minimal exposure time of <15 ms was required for the inactivation of over 99% pathogens in the aerosol; this is a key element in the design of a device that is safe and can be used in preventing inter-individual transmission. This represents a major advantage over existing devices, which mainly aim at either purifying incoming air by filters or sterilizing solid surfaces, which are not the major transmission routes for airborne communicable diseases

Flt1 produced by lung endothelial cells impairs ATII cell transdifferentiation and repair in pulmonary fibrosis

Pulmonary fibrosis is a devastating disease, in which fibrotic tissue progressively replaces lung alveolar structure, resulting in chronic respiratory failure. Alveolar type II cells act as epithelial stem cells, being able to transdifferentiate into alveolar type I cells, which mediate gas exchange, thus contributing to lung homeostasis and repair after damage. Impaired epithelial transdifferentiation is emerging as a major pathogenetic mechanism driving both onset and progression of fibrosis in the lung. Here, we show that lung endothelial cells secrete angiocrine factors that regulate alveolar cell differentiation. Specifically, we build on our previous data on the anti-fibrotic microRNA-200c and identify the Vascular Endothelial Growth Factor receptor 1, also named Flt1, as its main functional target in endothelial cells. Endothelial-specific knockout of Flt1 reproduces the anti-fibrotic effect of microRNA-200c against pulmonary fibrosis and results in the secretion of a pool of soluble factors and matrix components able to promote epithelial transdifferentiation in a paracrine manner. Collectively, these data indicate the existence of a complex endothelial-epithelial paracrine crosstalk in vitro and in vivo and position lung endothelial cells as a relevant therapeutic target in the fight against pulmonary fibrosis

Ischemic wound revascularization by the stromal vascular fraction relies on host-donor hybrid vessels

Nonhealing wounds place a significant burden on both quality of life of affected patients and health systems. Skin substitutes are applied to promote the closure of nonhealing wounds, although their efficacy is limited by inadequate vascularization. The stromal vascular fraction (SVF) from the adipose tissue is a promising therapy to overcome this limitation. Despite a few successful clinical trials, its incorporation in the clinical routine has been hampered by their inconsistent results. All these studies concluded by warranting pre-clinical work aimed at both characterizing the cell types composing the SVF and shedding light on their mechanism of action. Here, we established a model of nonhealing wound, in which we applied the SVF in combination with a clinical-grade skin substitute. We purified the SVF cells from transgenic animals to trace their fate after transplantation and observed that it gave rise to a mature vascular network composed of arteries, capillaries, veins, as well as lymphatics, structurally and functionally connected with the host circulation. Then we moved to a human-in-mouse model and confirmed that SVF-derived endothelial cells formed hybrid human-mouse vessels, that were stabilized by perivascular cells. Mechanistically, SVF-derived endothelial cells engrafted and expanded, directly contributing to the formation of new vessels, while a population of fibro-adipogenic progenitors stimulated the expansion of the host vasculature in a paracrine manner. These data have important clinical implications, as they provide a steppingstone toward the reproducible and effective adoption of the SVF as a standard care for nonhealing wounds

Studio del cross-talk tra cardiomiociti e cellule endoteliali per promuovere la rivascolarizzazione e la rigenerazione cardiaca

Il cuore adulto non è in grado di rigenerarsi o rivascolarizzarsi dopo un danno ischemico. Nell'ultimo decennio sono state prese in considerazione diverse strategie per sostituire i cardiomiociti (CM) persi o per promuovere la proliferazione di CM autologhi. Purtroppo, nessuno di questi approcci ha raggiunto con successo la fase clinica. Recenti studi suggeriscono il ruolo importante delle cellule endoteliali (EC), come regolatori chiave sia dell'omeostasi tissutale che della rigenerazione in diversi organi, come polmone, fegato, pancreas e muscolo. A differenza del cuore neonatale, il cuore adulto non è in grado di rivascolarizzarsi dopo un danno ischemico né dopo stimoli pro-angiogenici. Attualmente, sono disponibili poche informazioni sui meccanismi che bloccano l'angiogenesi nel cuore adulto e si sa molto poco sull'interazione EC-CM. Questo progetto intende indagare se i CM, che perdono il loro potenziale proliferativo una settimana dopo la nascita, inviano segnali inibitori che bloccano anche il potenziale angiogenico delle EC cardiache adulte. Utilizzando diversi metodi di co-coltura, abbiamo osservato che i CM adulti inviano segnali inibitori, che bloccano la proliferazione delle EC cardiache, mentre i CM neonatali non lo fanno. Per identificare i fattori anti-angiogenici, abbiamo sfruttato dati di sequenziamento dell'RNA per generare un interattoma che collega i ligandi prodotti dai CM con i recettori espressi dalle EC cardiache. Abbiamo osservato che 20 ligandi dei CM stabiliscono 118 possibili interazioni con 74 recettori delle EC. Approcci complementari di studi di perdita e guadagno di funzione hanno portato all'identificazione di potenziali interattori anti-angiogenici. Il knockout degli interattori ha ripristinato il potenziale angiogenico del cuore in risposta al VEGF. Partendo dall'osservazione che i CM adulti, ma non i neonatali, inibiscono la proliferazione delle EC, abbiamo testato se promuovere il parziale de-differenziazione dei CM potesse a sua volta ripristinare il potenziale angiogenico del cuore adulto. Pertanto, abbiamo somministrato contestualmente il miR199a, il miRNA con capacità pro-proliferative più potente, in combinazione con il VEGF-A utilizzando vettori virali adeno-associati. A seguito del co-trattamento abbiamo osservato una significativa proliferazione delle EC e la formazione di nuovi vasi sanguigni, confermando che il parziale de-differenziazione dei CM fa riacquistare il potenziale angiogenico alle EC cardiache. Una terza ipotesi per spiegare il basso potenziale angiogenico del cuore adulto è che l'endotelio cardiaco subisca modifiche cellulari autonome che riducono la sua capacità di rispondere a stimoli pro-angiogenici durante lo sviluppo postnatale. Tra i fattori che potrebbero essere coinvolti in questo processo, il recettore-1 del VEGF (VEGFR-1) è un candidato promettente, in quanto è noto inibire l'angiogenesi agendo come una molecola "esca" che impedisce il legame del VEGF-A al principale recettore angiogenico VEGFR-2. Abbiamo osservato che l'espressione di VEGFR-1 aumenta con l'età nelle EC, nelle CM e nelle cellule immunitarie. Per indagare il possibile ruolo di VEGFR-1 nella regolazione dell'angiogenesi cardiaca, abbiamo generato tre topi geneticamente modificati in cui abbiamo eliminato VEGFR-1 rispettivamente nelle EC, nelle CM e nelle cellule immunitarie residenti del cuore. A seguito della sovraespressione di VEGF, il knock-out di VEGFR-1 nelle EC, ma non nei CM e nelle cellule immunitarie residenti, ha aumentato l'angiogenesi cardiaca. Questi risultati confermano il ruolo cellulare-autonomo di VEGFR-1 nell'inibire la proliferazione delle EC nel cuore adulto. Questo lavoro ha fatto luce su tre meccanismi non mutuamente esclusivi che potrebbero essere responsabili del basso potenziale angiogenico del cuore adulto e che potrebbero essere sfruttati per sviluppare terapie innovative per favorire la rigenerazione e rivascolarizzazione cardiaca.The adult heart is not able to regenerate or revascularize itself after ischemic damage. In the past decade, several strategies have been considered to either replace lost cardiomyocytes (CMs) or promote autologous CM proliferation, unfortunately, none of these approaches have successfully reached the clinical phase. Recent observations suggest the important role of endothelial cells (ECs), as key regulators of both tissue homeostasis and regeneration in several organs, such as lung, liver, pancreas and muscle. Unlike the neonatal heart, the adult heart is not able to revascularize itself after ischemic damage neither after pro-angiogenic stimuli. Currently, scant information is available on the mechanisms blocking angiogenesis in the adult heart and very little is known about the EC-CM interaction. Because of their close proximity, these cells can communicate through either the secretion of paracrine signals or direct cell-to-cell contact. While several studies indicate that ECs provide multiple autocrine and paracrine signals controlling cardiac function, such as myocardial growth and CM contractility and homeostasis, much less is known about the signals produced by CMs, able to regulate EC biology. This project intends to investigate whether CMs, which lose their proliferation potential one week after birth, send inhibitory signals that also block the angiogenic potential of adult cardiac ECs. Using different co-culture methods, we observed that adult CMs indeed sent inhibitory signals, that blocked cardiac EC proliferation, whereas neonatal CMs did not. To identify these anti-angiogenic factors, we exploited existing RNA sequencing dataset to generate the interactome between ligands produced by CMs and receptors expressed by cardiac ECs. We observed that 20 CM ligands establish 118 putative interactions with 74 EC receptors. Complementary approaches of loss and gain of function studies led to identification of potentially anti-angiogenic interactors. The knockout of CM interactors rescued the angiogenic potential of the heart in response to VEGF. Thus, interfering with CM-EC cross-talk might offer new therapeutic avenues to promote cardiac angiogenesis. Starting from the observation that adult, but not neonatal, CMs inhibit EC proliferation, we explored whether partial CM de-differentiation could in turn rescue the angiogenic potential of the adult heart. Therefore, we contextually delivered miR199a, the most potent pro-proliferative miRNA, in combination with VEGF-A using adeno-associated viral vectors. This results in significant EC proliferation and neo-vessel formation, confirming that partial CM de-differentiation unleashes the angiogenic potential of cardiac ECs. A third hypothesis to explain the low angiogenic potential of the adult heart is that the cardiac endothelium undergoes cell autonomous modifications that reduce its capacity to respond to pro-angiogenic stimuli during post-natal development. Among the factors possibly involved in this process, VEGF receptor-1 (VEGFR-1) is a promising candidate acting as a "decoy" molecule that prevents the binding of VEGF-A to the main angiogenic receptor VEGFR-2. We observed that VEGFR-1 expression increases with age in cardiac ECs, CMs and immune cells. To investigate the possible role of VEGFR-1 in regulating cardiac angiogenesis, we generated three genetically modified mice in which we knocked-out VEGFR-1 in ECs, CMs and resident immune cells respectively. Upon VEGF overexpression, VEGFR-1 knock out in ECs, but not in both CMs and resident immune cells, increased cardiac angiogenesis. These results confirm the cell-autonomous role of VEGFR-1 in inhibiting EC proliferation in the heart. Collectively, this work has shed light on three non-mutually exclusive mechanisms that may be responsible for the low angiogenic potential of the adult heart and could be exploited to develop innovative therapies to foster cardiac regeneration and revascularization

Extracellular Matrix-Based Approaches in Cardiac Regeneration: Challenges and Opportunities

Cardiac development is characterized by the active proliferation of different cardiac cell types, in particular cardiomyocytes and endothelial cells, that eventually build the beating heart. In mammals, these cells lose their regenerative potential early after birth, representing a major obstacle to our current capacity to restore the myocardial structure and function after an injury. Increasing evidence indicates that the cardiac extracellular matrix (ECM) actively regulates and orchestrates the proliferation, differentiation, and migration of cardiac cells within the heart, and that any change in either the composition of the ECM or its mechanical properties ultimately affect the behavior of these cells throughout one’s life. Thus, understanding the role of ECMs’ proteins and related signaling pathways on cardiac cell proliferation is essential to develop effective strategies fostering the regeneration of a damaged heart. This review provides an overview of the components of the ECM and its mechanical properties, whose function in cardiac regeneration has been elucidated, with a major focus on the strengths and weaknesses of the experimental models so far exploited to demonstrate the actual pro-regenerative capacity of the components of the ECM and to translate this knowledge into new therapies

C1q as pro-angiogenic factor in the context of ovarian tissue transplantation

Problem: Anti-cancer therapy is increasingly effective, nonetheless, in women, it can result in iatrogenic premature ovarian insufficiency. In fact, follicle depletion is triggered by chemotherapy which induces apoptosis, interferes with cellular pathways responsible for quiescence maintenance, and damages the vascular network. For pre-pubertal girls and young women needing immediate therapy, the only option for fertility preservation is ovarian tissue cryopreservation and transplantation. A large part of follicle reserve is lost as a consequence of hypoxia due to an incomplete graft revascularization [1,2]. Ovarian tissue transplantation supplemented with autologous endothelial cells from ovary could result in a successful engraftment. Since previous work [3] already demonstrated that C1q could have a pro-angiogenic role, we decided to use this complement factor to enhance revascularization in this context. Method of Study: OVarian Endothelial Cells (OVECs) were isolated from ovarian biopsies from patients undergoing ovariectomy/annessiectomy. Tissue was digested by trypsin and, subsequently, by collagenase type I. Purification was achieved with a mixture of Dynabeads® CD31 and UEA-lectin (Ulex Europeus Aglutinine-Lectin)-conjugated beads. Angiogenic assays were performed in presence of C1q. The migration assay was conducted in a transwell system seeding cells in the upper chamber; the lower chamber was loaded with C1q and the percentage of migrated cells was calculated. In wound healing assay the monolayer of cells was stripped and C1q was added; the percentage of wound closure was estimated. For the tube formation assay cells were seeded in Matrigel® in presence of C1q; the following day, tubes were counted. We performed RT-qPCR to evaluate the expression of genes involved in the angiogenic process (VEGF-A, PlGF, ANGPT1, KDR, FLT-1, and TEK) in C1q-stimulated cells. Results: OVECs were characterized by immunofluorescence and cytofluorimetric analysis for panendothelial markers and cells resulted 95% positive for endothelial markers and negative for epithelial markers, indicating that we isolated an almost pure population. Both migration and wound healing assays indicated that C1q was able to induce a pro-migratory phenotype and enhanced OVECs’ ability to form capillary-like structures. To understand whether C1q was able to modulate the angiogenic process at gene level, RT-qPCR was performed onto C1q-treated cells. Results showed that it was able to increase the gene expression of the main pro-angiogenic factors and their receptors. Conclusions We were able to isolate an almost pure endothelial population from ovarian biopsies and C1q could induce a pro-angiogenic phenotype onto OVECs

EMID2 is a novel biotherapeutic for aggressive cancers identified by in vivo screening

Background: New drugs to tackle the next pathway or mutation fueling cancer are constantly proposed, but 97% of them are doomed to fail in clinical trials, largely because they are identified by cellular or in silico screens that cannot predict their in vivo effect. Methods: We screened an Adeno-Associated Vector secretome library (> 1000 clones) directly in vivo in a mouse model of cancer and validated the therapeutic effect of the first hit, EMID2, in both orthotopic and genetic models of lung and pancreatic cancer. Results: EMID2 overexpression inhibited both tumor growth and metastatic dissemination, consistent with prolonged survival of patients with high levels of EMID2 expression in the most aggressive human cancers. Mechanistically, EMID2 inhibited TGFβ maturation and activation of cancer-associated fibroblasts, resulting in more elastic ECM and reduced levels of YAP in the nuclei of cancer cells. Conclusion: This is the first in vivo screening, precisely designed to identify proteins able to interfere with cancer cell invasiveness. EMID2 was selected as the most potent protein, in line with the emerging relevance of the tumor extracellular matrix in controlling cancer cell invasiveness and dissemination, which kills most of cancer patients

Additional file 1 of EMID2 is a novel biotherapeutic for aggressive cancers identified by in vivo screening

Supplementary Material 1: Table S1. Composition of the pilot AAV9 poo