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Ischemia with non-obstructive coronary artery (INOCA) is a common cause of hospital admissions, leading to negative outcomes and reduced quality of life. Central to its pathophysiology is endothelial dysfunction, which contributes to myocardial ischemia despite the absence of significant coronary artery blockage. Addressing endothelial dysfunction is essential in managing INOCA to alleviate symptoms and prevent cardiovascular events. Recent studies have identified diabetes mellitus (DM) as a significant factor exacerbating INOCA complications by promoting endothelial impairment and coronary microvascular dysfunction. MicroRNAs (miRNAs) have emerged as potential biomarkers and therapeutic targets in various biological processes, including endothelial dysfunction and cardiovascular diseases. However, research on miRNA biomarkers in INOCA patients is sparse. In this study, we examined a panel of circulating miRNAs involved in the regulation of endothelial function in INOCA patients with and without DM. We analyzed miRNA expression using RT-qPCR in a cohort of consecutive INOCA patients undergoing percutaneous coronary intervention. We detected a significant dysregulation of miR-363-5p and miR-92a-3p in INOCA patients with DM compared to those without DM, indicating their role as biomarkers for predicting and monitoring endothelial dysfunction in INOCA patients with DM., (© 2024. The Author(s).)

Emerging evidence indicates that the relationship between coronavirus disease 2019 (COVID-19) and diabetes is 2-fold: 1) it is known that the presence of diabetes and other metabolic alterations poses a considerably high risk to develop a severe COVID-19; 2) patients who survived a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection have an increased risk of developing new-onset diabetes. However, the mechanisms underlying this association are mostly unknown, and there are no reliable biomarkers to predict the development of new-onset diabetes. In the present study, we demonstrate that a specific microRNA (miR-34a) contained in circulating extracellular vesicles released by endothelial cells reliably predicts the risk of developing new-onset diabetes in COVID-19. This association was independent of age, sex, body mass index (BMI), hypertension, dyslipidemia, smoking status, and D-dimer. SIGNIFICANCE STATEMENT: We demonstrate for the first time that a specific microRNA (miR-34a) contained in circulating extracellular vesicles released by endothelial cells is able to reliably predict the risk of developing diabetes after having contracted coronavirus disease 2019 (COVID-19). This association was independent of age, sex, body mass index (BMI), hypertension, dyslipidemia, smoking status, and D-dimer. Our findings are also relevant when considering the emerging importance of post-acute sequelae of COVID-19, with systemic manifestations observed even months after viral negativization (long COVID)., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

Background: Cardiac fibrosis represents a key element in the pathophysiology of heart failure with preserved ejection fraction (HFpEF), a condition highly prevalent amongst geriatric patients, especially if diabetic. The microRNA 181c (miR-181c) has been shown to be associated with the response to exercise training in HFpEF patients and has been also linked to diabetic cardiovascular complications. However, the underlying mechanisms have not been fully elucidated., Objective: To measure circulating miR-181c in elderly patients with HFpEF and diabetes mellitus (DM) and identify gene targets pathophysiologically relevant in HFpEF., Methods: We quantified circulating miR-181c in frail older adults with a confirmed diagnosis of HFpEF and DM, and, as control, we enrolled age-matched subjects without HFpEF and without DM. We validated in human cardiac fibroblasts the molecular mechanisms linking miR-181c to a pro-fibrotic response., Results: 51 frail patients were included :34 patients with DM and HFpEF and 17 age-matched controls. We observed that miR-181c was significantly upregulated (p < 0.0001) in HFpEF patients vs controls. We confirmed in vitro that miR-181c is targeting PRKN and SMAD7., Conclusions: We demonstrate that miR-181c levels are significantly increased in frail elderly adults with DM and HFpEF and that miR-181c targets PRKN and SMAD7 in human cardiac fibroblasts., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Endothelial dysfunction (ED) causes worse prognoses in heart failure (HF) patients treated with cardiac resynchronization therapy (CRTd). ED triggers the downregulation of microRNA-130 (miR-130a-5p), which targets endothelin-1 (ET-1). Thus, we evaluated ED and the response to CRTd by assessing miR-130a-5p and ET-1 serum levels. We designed a prospective multi-center study with a 1-year follow-up to evaluate ED, ET-1, and miR-130a-5p in CRTd patients with ED (ED-CRTd) vs. patients without ED (NED-CRTd). Clinical outcomes were CRTd response, HF hospitalization, cardiac death, and all-cause death. At 1-year follow-up, NED-CRTd (n = 541) vs. ED-CRTd (n = 326) patients showed better clinical statuses, lower serum values of B type natriuretic peptide (BNP: 266.25 ± 10.8 vs. 297.43 ± 16.22 pg/mL; p < 0.05) and ET-1 (4.57 ± 0.17 vs. 5.41 ± 0.24 pmol/L; p < 0.05), and higher values of miR-130a-5p (0.51 ± 0.029 vs. 0.41 ± 0.034 A.U; p < 0.05). Compared with NED-CRTd patients, ED-CRTd patients were less likely to be CRTd responders (189 (58%) vs. 380 (70.2%); p < 0.05) and had higher rates of HF hospitalization (115 (35.3%) vs. 154 (28.5%); p < 0.05) and cardiac deaths (30 (9.2%) vs. 21 (3.9%); p < 0.05). Higher miR-130a-5p levels (HR 1.490, CI 95% [1.014−2.188]) significantly predicted CRTd response; the presence of hypertension (HR 0.818, CI 95% [0.669−0.999]), and displaying higher levels of ET-1 (HR 0.859, CI 98% [0.839−0.979]), lymphocytes (HR 0.820, CI 95% [0.758−0.987]), LVEF (HR 0.876, CI 95% [0.760−0.992]), and ED (HR 0.751, CI 95% [0.624−0.905]) predicted CRTd non-response. Higher serum miR-130a-5p levels (HR 0.332, CI 95% [0.347−0.804]) and use of ARNI (HR 0.319, CI 95% [0.310−0.572]) predicted lower risk of HF hospitalization, whereas hypertension (HR 1.818, CI 95% [1.720−2.907]), higher BNP levels (HR 1.210, CI 95% [1.000−1.401]), and presence of ED (HR 1.905, CI 95% [1.238−2.241]) predicted a higher risk of HF hospitalization. Hence, serum miR-130a-5p could identify different stages of ED and independently predict CRTd response, therefore representing a novel prognostic HF biomarker.

We hypothesized that exosomal microRNAs could be implied in the pathogenesis of thromboembolic complications in coronavirus disease 2019 (COVID-19). We isolated circulating exosomes from patients with COVID-19, and then we divided our population in two arms based on the D-dimer level on hospital admission. We observed that exosomal miR-145 and miR-885 significantly correlate with D-dimer levels. Moreover, we demonstrate that human endothelial cells express the main cofactors needed for the internalization of the "Severe acute respiratory syndrome coronavirus 2" (SARS-CoV-2), including angiotensin converting enzyme 2, transmembrane protease serine 2, and CD-147. Interestingly, human endothelial cells treated with serum from COVID-19 patients release significantly less miR-145 and miR-885, exhibit increased apoptosis, and display significantly impaired angiogenetic properties compared with cells treated with non-COVID-19 serum. Taken together, our data indicate that exosomal miR-145 and miR-885 are essential in modulating thromboembolic events in COVID-19. SIGNIFICANCE STATEMENT: This work demonstrates for the first time that two specific microRNAs (namely miR-145 and miR-885) contained in circulating exosomes are functionally involved in thromboembolic events in COVID-19. These findings are especially relevant to the general audience when considering the emerging prominence of post-acute sequelae of COVID-19 systemic manifestations known as Long COVID., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

Endothelial dysfunction represents a key mechanism underlying heart failure with preserved ejection fraction (HFpEF), diabetes mellitus (DM), and frailty. However, reliable biomarkers to monitor endothelial dysfunction in these patients are lacking. In this study, we evaluated the expression of a panel of circulating microRNAs (miRs) involved in the regulation of endothelial function in a population of frail older adults with HFpEF and DM treated for 3 months with empagliflozin, metformin, or insulin. We identified a distinctive pattern of miRs that were significantly regulated in HFpEF patients compared to healthy controls and to HFpEF patients treated with the sodium glucose cotransporter 2 (SGLT2) inhibitor empagliflozin. Three miRs were significantly downregulated (miR-126, miR-342-3p, and miR-638) and two were significantly upregulated (miR-21 and miR-92) in HFpEF patients compared to healthy controls. Strikingly, two of these miRs (miR-21 and miR-92) were significantly reduced in HFpEF patients after the 3-month treatment with empagliflozin, whereas no significant differences in the profile of endothelial miRs were detected in patients treated with metformin or insulin. Taken together, our findings demonstrate for the first time that specific circulating miRs involved in the regulation of endothelial function are significantly regulated in frail HFpEF patients with DM and in response to SGLT2 inhibition. SIGNIFICANCE STATEMENT: We have identified a novel microRNA signature functionally involved in the regulation of endothelial function that is significantly regulated in frail patients with HFpEF and diabetes. Moreover, the treatment with the SGLT2 inhibitor empagliflozin caused a modification of some of these microRNAs in a direction that was opposite to what observed in HFpEF patients, indicating a rescue of endothelial function. Our findings are relevant for clinical practice inasmuch as we were able to establish novel biomarkers of disease and response to therapy., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

Fabry disease (FD) is a lysosomal storage disorder caused by mutations in the gene for α -galactosidase A, inducing a progressive accumulation of globotriaosylceramide (GB3) and its metabolites in different organs and tissues. GB3 deposition does not fully explain the clinical manifestations of FD, and other pathogenetic mechanisms have been proposed, requiring the identification of new biomarkers for monitoring FD patients. Emerging evidence suggests the involvement of mitochondrial alterations in FD. Here, we propose mitochondrial-related microRNAs (miRs) as potential biomarkers of mitochondrial involvement in FD. Indeed, we demonstate that miRs regulating different aspects of mitochondrial homeostasis including expression and assembly of respiratory chain, mitogenesis, antioxidant capacity, and apoptosis are consistently dysregulated in FD patients. Our data unveil a novel noncoding RNA signature of FD patients, indicating mitochondrial-related miRs as new potential pathogenic players and biomarkers in FD. SIGNIFICANCE STATEMENT: This study demonstrates for the first time that a specific signature of circulating mitochondrial miRs (mitomiRs) is dysregulated in FD patients. MitomiRs regulating fundamental aspects of mitochondrial homeostasis and fitness, including expression and assembly of the respiratory chain, mitogenesis, antioxidant capacity, and apoptosis are significantly dysregulated in FD patients. Taken together, these new findings introduce mitomiRs as unprecedented biomarkers of FD and point at mitochondrial dysfunction as a novel potential mechanistic target for therapeutic approaches., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

T-cell immunoglobulin and mucin domain 1 (TIM-1) has been recently identified as one of the factors involved in the internalization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human cells, in addition to angiotensin-converting enzyme 2 (ACE2), transmembrane serine protease 2 (TMPRSS2), neuropilin-1, and others. We hypothesized that specific microRNAs could target TIM-1, with potential implications for the management of patients suffering from coronavirus disease 2019 (COVID-19). By combining bioinformatic analyses and functional assays, we identified miR-142 as a specific regulator of TIM-1 transcription. Since TIM-1 has been implicated in the regulation of endothelial function at the level of the blood-brain barrier (BBB) and its levels have been shown to be associated with stroke and cerebral ischemia-reperfusion injury, we validated miR-142 as a functional modulator of TIM-1 in human brain microvascular endothelial cells (hBMECs). Taken together, our results indicate that miR-142 targets TIM-1, representing a novel strategy against cerebrovascular disorders, as well as systemic complications of SARS-CoV-2 and other viral infections.

microRNAs (miRNA, miRs) play crucial roles in cardiovascular disease regulating numerous processes, including inflammation, cell proliferation, angiogenesis, and cell death. Herein, we present an updated and comprehensive overview of the functional involvement of miRs in the regulation of cardiomyocyte death, a central event in acute myocardial infarction, ischemia/reperfusion, and heart failure. Specifically, in this systematic review we are focusing on necrosis, apoptosis, and autophagy.

Glucagon-like peptide-1 (GLP-1) has been shown to potentiate glucose-stimulated insulin secretion binding GLP-1 receptor on pancreatic β cells. β-arrestin 1 (βARR1) is known to regulate the desensitization of GLP-1 receptor. Mounting evidence indicates that microRNAs (miRNAs, miRs) are fundamental in the regulation of β cell function and insulin release. However, the regulation of GLP-1/βARR1 pathways by miRs has never been explored. Our hypothesis is that specific miRs can modulate the GLP-1/βARR1 axis in β cells. To test this hypothesis, we applied a bioinformatic approach to detect miRs that could target βARR1; we identified hsa-miR-7-5p (miR-7) and we validated the specific interaction of this miR with βARR1. Then, we verified that GLP-1 was indeed able to regulate the transcription of miR-7 and βARR1, and that miR-7 significantly regulated GLP-1-induced insulin release and cyclic AMP (cAMP) production in β cells. Taken together, our findings indicate, for the first time, that miR-7 plays a functional role in the regulation of GLP-1-mediated insulin release by targeting βARR1. These results have a decisive clinical impact given the importance of drugs modulating GLP-1 signaling in the treatment of patients with type 2 diabetes mellitus.

Aims: We hypothesize that specific microRNAs (miRNAs) within cardiomyocyte-derived exosomes play a pivotal role in the phenoconversion of cardiac myofibroblasts following myocardial infarction (MI)., Methods and Results: We used an established murine model of MI, obtained in vivo via ligation of the left anterior descending coronary artery. We isolated adult cardiomyocytes and fibroblasts, and we assessed the functional role of cardiomyocyte-derived exosomes and their molecular cargo in the activation of cardiac fibroblasts. We identified and biologically validated miR-92a as a transcriptional regulator of mothers against DPP homologues 7 (SMAD7), a known inhibitor of α-smooth muscle actin (α-SMA), established marker of myofibroblast activation. We found that miR-92a was significantly (P < 0.05) upregulated in cardiomyocyte-derived exosomes and in fibroblasts isolated after MI compared with SHAM conditions (n ≥ 6/group). We tested the activation of myofibroblasts by measuring the expression levels of αSMA, periostin, and collagen. Primary isolated cardiac fibroblasts were activated both when incubated with cardiomyocyte-derived exosomes isolated from ischemic cardiomyocytes and when cultured in conditioned medium of post-MI cardiomyocytes, whereas no significant difference was observed following incubation with exosomes or medium from sham cardiomyocytes. These effects were attenuated when an inhibitor of exosome secretion, GW4869 (10 μM for 12 h) was included in the experimental setting. Through means of specific miR-92a mimic and miR-92a inhibitor, we also verified the mechanistic contribution of miR-92a to the activation of cardiac fibroblasts., Conclusions: Our results indicate for the first time that miR-92a is transferred to fibroblasts in form of exosomal cargo and is critical for cardiac myofibroblast activation., (© 2020 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of the European Society of Cardiology.)

The inclusion of microRNAs (miRNAs) in extracellular microvesicles/exosomes (named cardiosomes when deriving from cardiomyocytes) allows their active transportation and ensures cell-cell communication. We hypothesize that cardiosomal miRNAs play a pivotal role in the activation of myofibroblasts following ischemic injury. Using a murine model of myocardial infarction (MI), we tested our hypothesis by measuring in isolated fibroblasts and cardiosomes the expression levels of a set of miRNAs, which are upregulated in cardiomyocytes post-MI and involved in myofibroblast phenoconversion. We found that miR-195 was significantly upregulated in cardiosomes and in fibroblasts isolated after MI compared with SHAM conditions. Moreover, primary isolated cardiac fibroblasts were activated both when incubated with cardiosomes isolated from ischemic cardiomyocytes and when cultured in conditioned medium of post-MI cardiomyocytes, whereas no significant effect was observed following incubation with cardiosomes or medium from sham cardiomyocytes. Taken together, our findings indicate for the first time that a cardiomyocyte-specific miRNA, transferred to fibroblasts in form of exosomal cargo, is crucial in the activation of myofibroblasts.

T lymphocytes release exosomes containing specific microRNAs that cause apoptosis of pancreatic β cells in type 1 diabetes.

Accumulating evidence indicates that microRNAs (miRs)-non-coding RNAs that can regulate gene expression via translational repression and/or post-transcriptional degradation-are becoming one of the most fascinating areas of physiology, given their fundamental roles in countless pathophysiological processes. The relative roles of different miRs in vascular biology as direct or indirect post-transcriptional regulators of fundamental genes implied in vascular remodeling designate miRs as potential biomarkers and/or promising drug targets. The mechanistic importance of miRs in modulating endothelial cell (EC) function in physiology and in disease is addressed here. Drawbacks of currently available therapeutic options are also discussed, pointing at the challenges and clinical opportunities provided by miR-based treatments. J. Cell. Physiol. 231: 1638-1644, 2016. © 2015 Wiley Periodicals, Inc., (© 2015 Wiley Periodicals, Inc.)

MicroRNAs (miRs) are important regulators of gene expression in numerous biological processes. Their maturation process is herein described, including the most updated insights from the current literature. Circa 2000 miR sequences have been identified in the human genome, with over 50,000 miR-target interactions, including enzymes involved in epigenetic modulation of gene expression. Moreover, some "pieces of RNA" previously annotated as noncoding have been recently found to encode micropeptides that carry out critical mechanistic functions in the cell. Advanced techniques now available will certainly allow a precise scanning of the genome looking for micropeptides hidden within the "noncoding" RNA.

MicroRNAs (miRNAs, miRs) represent a group of powerful and versatile posttranscriptional regulators of gene expression being involved in the fine control of a plethora of physiological and pathological processes. Besides their well-established crucial roles in the regulation of cell cycle, embryogenesis or tumorigenesis, these tiny molecules have also been shown to participate in the regulation of lipid metabolism. In particular, miRs orchestrate cholesterol and fatty acids synthesis, transport, and degradation and low-density and high-density lipoprotein (LDL and HDL) formation. It is thus not surprising that they have also been reported to affect the development and progression of several lipid metabolism-related disorders including liver steatosis and atherosclerosis. Mounting evidence suggests that miRs might represent important "posttranscriptional hubs" of lipid metabolism, which means that one miR usually targets 3'-untranslated regions of various mRNAs that are involved in different steps of one precise metabolic/signaling pathway, e.g., one miR targets mRNAs of enzymes important for cholesterol synthesis, degradation, and transport. Therefore, changes in the levels of one key miR affect various steps of one pathway, which is thereby promoted or inhibited. This makes miRs potent future diagnostic and even therapeutic tools for personalized medicine. Within this chapter, the most prominent microRNAs involved in lipid metabolism, e.g., miR-27a/b, miR-33/33*, miR-122, miR-144, or miR-223, and their intracellular and extracellular functions will be extensively discussed, in particular focusing on their mechanistic role in the pathophysiology of atherosclerosis. Special emphasis will be given on miR-122, the first microRNA currently in clinical trials for the treatment of hepatitis C and on miR-223, the most abundant miR in lipoprotein particles.

Endothelial cells (EC) and vascular smooth muscle cells (VSMC) are the main cell types within the vasculature. We describe here how microRNAs (miRs)--noncoding RNAs that can regulate gene expression via translational repression and/or post-transcriptional degradation--distinctively modulate EC and VSMC function in physiology and disease. In particular, the specific roles of miR-126 and miR-143/145, master regulators of EC and VSMC function, respectively, are deeply explored. We also describe the mechanistic role of miRs in the regulation of the pathophysiology of key cardiovascular processes including angiogenesis, atherosclerosis, and in-stent restenosis post-angioplasty. Drawbacks of currently available therapeutic options are discussed, pointing at the challenges and potential clinical opportunities provided by miR-based treatments.

In his State of the Union address before both chambers of the US Congress, President Barack Obama called for increased investment in US infrastructure and research and announced the launch of a new Precision Medicine Initiative, aiming to accelerate biomedical discovery. Due to their well-established selectivity and specificity, microRNAs can represent a useful tool, both in diagnosis and therapy, in forging the path towards the achievement of precision medicine. This introductory chapter represents a guide for the Reader in examining the functional roles of microRNAs in the most diverse aspects of clinical practice, which will be explored in this third volume of the microRNA trilogy.

Cardiovascular disease (CVD) is a major cause of morbidity and mortality worldwide. Innovative, more stringent diagnostic and prognostic biomarkers and effective treatment options are needed to lessen its burden. In recent years, microRNAs have emerged as master regulators of gene expression - they bind to complementary sequences within the mRNAs of their target genes and inhibit their expression by either mRNA degradation or translational repression. microRNAs have been implicated in all major cellular processes, including cell cycle, differentiation and metabolism. Their unique mode of action, fine-tuning gene expression rather than turning genes on/off, and their ability to simultaneously regulate multiple elements of relevant pathways makes them enticing potential biomarkers and therapeutic targets. Indeed, cardiovascular patients have specific patterns of circulating microRNA levels, often early in the disease process. This article provides a systematic overview of the role of microRNAs in the pathophysiology, diagnosis and treatment of CVD., (© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Drugs currently approved to coat stents used in percutaneous coronary interventions do not discriminate between proliferating vascular smooth muscle cells (VSMCs) and endothelial cells (ECs). This lack of discrimination delays reendothelialization and vascular healing, increasing the risk of late thrombosis following angioplasty. We developed a microRNA-based (miRNA-based) approach to inhibit proliferative VSMCs, thus preventing restenosis, while selectively promoting reendothelialization and preserving EC function. We used an adenoviral (Ad) vector that encodes cyclin-dependent kinase inhibitor p27(Kip1) (p27) with target sequences for EC-specific miR-126-3p at the 3' end (Ad-p27-126TS). Exogenous p27 overexpression was evaluated in vitro and in a rat arterial balloon injury model following transduction with Ad-p27-126TS, Ad-p27 (without miR-126 target sequences), or Ad-GFP (control). In vitro, Ad-p27-126TS protected the ability of ECs to proliferate, migrate, and form networks. At 2 and 4 weeks after injury, Ad-p27-126TS-treated animals exhibited reduced restenosis, complete reendothelialization, reduced hypercoagulability, and restoration of the vasodilatory response to acetylcholine to levels comparable to those in uninjured vessels. By incorporating miR-126-3p target sequences to leverage endogenous EC-specific miR-126, we overexpressed exogenous p27 in VSMCs, while selectively inhibiting p27 overexpression in ECs. Our proof-of-principle study demonstrates the potential of using a miRNA-based strategy as a therapeutic approach to specifically inhibit vascular restenosis while preserving EC function.

Heart failure (HF) disease progression is related to numerous adaptive processes including cardiac fibrosis, hypertrophy and apoptosis by activation of the 'fetal' gene program and downregulation of mRNA signatures, suggesting the importance of molecular mechanisms that suppress mRNA steady-state levels. miRNAs (miRs) are small, noncoding RNAs that bind mRNAs at their 3'-UTRs, leading to mRNA degradation or inhibition of protein translation. Several miRs are unregulated in response to cellular stress and can modify cellular functions such as proliferation, differentiation and programmed death; these miRs are also regulated in cardiac disease. Cardiac resynchronization therapy improves cardiac performance and myocardial mechanical efficiency. In this updated critical appraisal we report on the main miRs that play a key role in response to cardiac resynchronization therapy (i.e., responder vs nonresponder HF patients), focusing on the miR-mediated modulation of cardiac angiogenesis, apoptosis, fibrosis and membrane ionic currents.

The mTOR signaling pathway integrates inputs from a variety of upstream stimuli to regulate diverse cellular processes including proliferation, growth, survival, motility, autophagy, protein synthesis and metabolism. The mTOR pathway is dysregulated in a number of human pathologies including cancer, diabetes, obesity, autoimmune disorders, neurological disease and aging. Ongoing clinical trials testing mTOR-targeted treatments number in the hundreds and underscore its therapeutic potential. To date mTOR inhibitors are clinically approved to prevent organ rejection, to inhibit restenosis after angioplasty, and to treat several advanced cancers. In this review we discuss the continuously evolving field of mTOR pharmacogenomics, as well as highlight the emerging efforts in identifying diagnostic and prognostic markers, including miRNAs, in order to assess successful therapeutic responses.