Periodic Reporting for period 1 - CVD in RA (Mechanistic links between rheumatoid arthritis and cardiovascular complications: investigation on inflammation induced alterations in induced pluripotent stem cell-derived cardiomyocytes)
Periodo di rendicontazione: 2021-03-01 al 2023-02-28
Rheumatoid arthritis (RA) is a chronic and progressive inflammatory disease of the joints characterized by synovial inflammation and destruction of cartilage and bone. There is increasing awareness that RA is not only limited to the local joint inflammatory state. Of emerging significance is the relationship between RA and systemic inflammation which increases damage to cardiac and endothelial cells. This is coupled with impaired heart and vascular functions, thus contributing to the risk of cardiovascular disease (CVD). Subsequently, CVD is the main cause of the excess morbidity and mortality risk as RA patients may have cardiac problems within one year following diagnosis. Moreover, the risk of having a heart attack and other forms of coronary heart disease increases by 60% in one to four years after the RA diagnosis. Although this recognition has been described by clinicians, the exact reason for the increased CV risk in RA patients is still under intense study. In addition, a novel biological medication used to treat RA may modulate CV risk, therefore should be addressed in cardiovascular drug toxicity. For this reason, the main objective of this project was to generate a novel cellular tool that may help to identify more specific molecular mechanisms associated with the development of heart complications in RA patients.
To date, most of the RA research has been traditionally based on a variety of in vitro assays and animal models which may not entirely recapitulate extra-articular manifestations and drug responses. In turn, human cardiomyocytes are difficult to access from the heart of RA patients. Therefore, in this project, we implemented the modern technology of induced pluripotent stem cells (iPSC), pioneered by Shinya Yamanaka’s lab in Kyoto, Japan, who was awarded the 2012 Nobel Prize. iPSCs are a type of stem cell that can be generated directly from adult cells like fibroblasts or blood cells. iPSCs have the same properties as embryonic stem cells, i.e. self-renewing and pluripotent differentiation giving rise to many other cell types, such as neurons, heart, pancreatic, and liver cells. Therefore, nowadays iPSCs have become an important tool for modeling and investigating many human diseases, for screening drugs, and in the field of regenerative medicine.
To date, most of the RA research has been traditionally based on a variety of in vitro assays and animal models which may not entirely recapitulate extra-articular manifestations and drug responses. In turn, human cardiomyocytes are difficult to access from the heart of RA patients. Therefore, in this project, we implemented the modern technology of induced pluripotent stem cells (iPSC), pioneered by Shinya Yamanaka’s lab in Kyoto, Japan, who was awarded the 2012 Nobel Prize. iPSCs are a type of stem cell that can be generated directly from adult cells like fibroblasts or blood cells. iPSCs have the same properties as embryonic stem cells, i.e. self-renewing and pluripotent differentiation giving rise to many other cell types, such as neurons, heart, pancreatic, and liver cells. Therefore, nowadays iPSCs have become an important tool for modeling and investigating many human diseases, for screening drugs, and in the field of regenerative medicine.
This project employed a truly translational, bench-to-bedside approach built on the strength of the human RA model. In collaboration with Rheumatologists, human samples of joint fibroblasts (FLS) and peripheral blood mononuclear cells (PBMC) were collected from healthy donors and RA patients. A reprogramming method with the Yamanaka cocktail (hOct3/4, hSox2, hKlf4, and hc-Myc) allowed us to successfully generate and characterize FLS-derived iPSCs and PBMC-derived iPSCs, which demonstrated typical colony-like morphology and presence of pluripotent markers.
Next, using the novel differentiation strategies, FLS-derived iPSCs and PBMC-derived iPSCs were converted into cardiac lineage differentiated cells, like cardiomyocytes (CM), endothelial cells (EC), and cardiac fibroblasts (CF). As the native cardiac tissue is composed of heterogeneous cell populations that work cooperatively for proper tissue functions, we have generated human cardiac microtissues (MT) in vitro composed of iPSC-CMs and non-myocytes (iPSC-EC and iPSC-CF) in complex 3D structure.
To imitate the impact of joint inflammation and medications on cardiomyocyte properties and functions, cells were stimulated with key RA pro-inflammatory cytokines and anti-RA drugs. Later, the state-of-the-art technologies were applied. Cell morphology and the presence of specific markers were examined by immunocytochemistry and captured by immunofluorescence microscopy. To measure the physical and chemical characteristics of a single cell, a technique of flow cytometry was used. The electrical activity of contracting cardiomyocytes was accessed by patch-clamp technology. Quantification of real-time bioenergetics was determined by the cell metabolism analyzer Seahorse. Expression of cell-specific genes was measured by polymerase chain reaction (PCR).
Applying the above techniques, we confirmed that all established in this project cell lines are functional and possess the desired cell-specific markers and properties. The preliminary data from this project suggests that RA pro-inflammatory cytokines change the electrophysiological and bioenergetics functions of cardiomyocytes, promoting CV complication in RA and this effect can be abrogated by the novel anti-RA drugs. Based on our initial conclusions, we will further identify cardiomyocyte-specific genes and pathways that might affect electrophysiology and cellular energy metabolism in RA.
Next, using the novel differentiation strategies, FLS-derived iPSCs and PBMC-derived iPSCs were converted into cardiac lineage differentiated cells, like cardiomyocytes (CM), endothelial cells (EC), and cardiac fibroblasts (CF). As the native cardiac tissue is composed of heterogeneous cell populations that work cooperatively for proper tissue functions, we have generated human cardiac microtissues (MT) in vitro composed of iPSC-CMs and non-myocytes (iPSC-EC and iPSC-CF) in complex 3D structure.
To imitate the impact of joint inflammation and medications on cardiomyocyte properties and functions, cells were stimulated with key RA pro-inflammatory cytokines and anti-RA drugs. Later, the state-of-the-art technologies were applied. Cell morphology and the presence of specific markers were examined by immunocytochemistry and captured by immunofluorescence microscopy. To measure the physical and chemical characteristics of a single cell, a technique of flow cytometry was used. The electrical activity of contracting cardiomyocytes was accessed by patch-clamp technology. Quantification of real-time bioenergetics was determined by the cell metabolism analyzer Seahorse. Expression of cell-specific genes was measured by polymerase chain reaction (PCR).
Applying the above techniques, we confirmed that all established in this project cell lines are functional and possess the desired cell-specific markers and properties. The preliminary data from this project suggests that RA pro-inflammatory cytokines change the electrophysiological and bioenergetics functions of cardiomyocytes, promoting CV complication in RA and this effect can be abrogated by the novel anti-RA drugs. Based on our initial conclusions, we will further identify cardiomyocyte-specific genes and pathways that might affect electrophysiology and cellular energy metabolism in RA.
This project allowed to generate of several unique patient-specific iPSCs lines used to examine the pathophysiology of cardiac involvement in RA. All generated cell lines are readily obtainable for third parties and can be further used for the scientific and clinical community i.e. for research projects involving differentiation of iPSCs into cells of tendon, cartilage, and bone, in order to develop cell-based joint regenerative therapies. Wide application of patient-specific iPSCs in arthritic research may lead to a better understanding and management of this debilitating disease and its relationship with cardiovascular comorbidity. This, in turn, will reduce individual, economic, and social costs, including drugs, hospitalizations, lost workdays, costs to family and careers, and an overall reduced quality of life.
Established in this project 3D model of cardiac microtissue can represent a valid in vitro model to investigate cardiac CM/EC/CF cellular dialogue not only in RA, but also in other chronic inflammatory diseases, such as colorectal disease, cancer, and diabetes. Subsequently, it may lead to the preclinical implementation of cardiac microtissues as a platform for precision medicine to evaluate drug cardioprotection or cardiotoxicity with a significant prognostic value and improvement of treatment management. Importantly, currently, the human-relevant iPSC-CMs platform is increasingly used in pre-clinical in vitro cardiac toxicity screening and has been accepted for investigational new drug (IND) applications from a clinical study sponsor to obtain authorization from the Food and Drug Administration (FDA).
Established in this project 3D model of cardiac microtissue can represent a valid in vitro model to investigate cardiac CM/EC/CF cellular dialogue not only in RA, but also in other chronic inflammatory diseases, such as colorectal disease, cancer, and diabetes. Subsequently, it may lead to the preclinical implementation of cardiac microtissues as a platform for precision medicine to evaluate drug cardioprotection or cardiotoxicity with a significant prognostic value and improvement of treatment management. Importantly, currently, the human-relevant iPSC-CMs platform is increasingly used in pre-clinical in vitro cardiac toxicity screening and has been accepted for investigational new drug (IND) applications from a clinical study sponsor to obtain authorization from the Food and Drug Administration (FDA).