RV-specific Targeting of Snai1 Rescues Pulmonary Hypertension-induced Right Ventricular Failure by Inhibiting EndMT and Fibrosis via LOXL2 Mediated Mechanism.

Background: Pulmonary hypertension (PH)-induced right ventricular (RV) failure (PH-RVF) is a significant prognostic determinant of mortality and is characterized by RV hypertrophy, endothelial-to-mesenchymal transition (EndMT), fibroblast-to-myofibroblast transition (FMT), fibrosis, and extracellular matrix (ECM)-remodeling. Despite the importance of RV function in PH, the mechanistic details of PH-RVF, especially the regulatory control of RV EndMT, FMT, and fibrosis, remain unclear. The action of transcription factor Snai1 is shown to be mediated through LOXL2 recruitment, and their co-translocation to the nucleus, during EndMT progression. We hypothesize that RV EndMT and fibrosis in PH-RVF are governed by the TGFß1-Snai1-LOXL2 axis. Furthermore, targeting Snai1 could serve as a novel therapeutic strategy for PH-RVF. Methods: Adult male Sprague Dawley rats (250-300g) received either a single subcutaneous injection of Monocrotaline (MCT, 60mg/kg, n=9; followed for 30-days) or Sugen (SU5416 20mg/kg, n=9; 10% O 2 hypoxia for 3-weeks followed by normoxia for 2-weeks) or PBS (CTRL, n=9). We performed secondary bioinformatics analysis on the RV bulk RNA-Seq data from MCT, SuHx, and PAB rats and human PH-PVF. We validated EndMT and FMT and their association with Snai1 and LOXL2 in the RVs of MCT and SuHx rat models and human PH-RVF using immunofluorescence, qPCR, and Western blots. For in vivo Snai1 knockdown (Snai1-KD), MCT-rats either received Snai1-siRNA (n=7; 5nM/injection every 3-4 days; 4-injections) or scramble (SCRM-KD; n=7) through tail vein from day 14-30 after MCT. Echocardiography and catheterization were performed terminally. Bulk RNASeq and differential expression analysis were performed on Snai1- and SCRM-KD rat RVs. In vitro Snai1-KD was performed on human coronary artery endothelial cells (HCAECs) and human cardiac fibroblasts (HCFs) under hypoxia+TGFß1 for 72-hrs. Results: PH-RVF had increased RVSP and Fulton index and decreased RV fractional area change (RVFAC %). RV RNASeq demonstrated EndMT as the common top-upregulated pathway between rat (MCT, SuHx, and PAB) and human PH-RVF. Immunofluorescence using EndMT- and FMT-specific markers demonstrated increased EndMT and FMT in RV of MCT and SuHx rats and PH-RVF patients. Further, RV expression of TGFß1, Snai1, and LOXL2 was increased in MCT and SuHx. Nuclear co-localization and increased immunoreactivity, transcript, and protein levels of Snai1 and LOXL2 were observed in MCT and SuHx rats and human RVs. MCT rats treated with Snai1-siRNA demonstrated decreased Snai1 expression, RVSP, Fulton index, and increased RVFAC. Snai1-KD resulted in decreased RV-EndMT, FMT, and fibrosis via a LOXL2-dependent manner. Further, Snai1-KD inhibited hypoxia+TGFß1-induced EndMT in HCAECs and FMT in HCFs in vitro by decreasing perinuclear/nuclear Snai1+LOXL2 expression and co-localization. Conclusions: RV-specific targeting of Snai1 rescues PH-RVF by inhibiting EndMT and Fibrosis via a LOXL2-mediated mechanism.

Deficiency of the Deubiquitinase UCHL1 Attenuates Pulmonary Arterial Hypertension.

BACKGROUND: The ubiquitin-proteasome system regulates protein degradation and the development of pulmonary arterial hypertension (PAH), but knowledge about the role of deubiquitinating enzymes in this process is limited. UCHL1 (ubiquitin carboxyl-terminal hydrolase 1), a deubiquitinase, has been shown to reduce AKT1 (AKT serine/threonine kinase 1) degradation, resulting in higher levels. Given that AKT1 is pathological in pulmonary hypertension, we hypothesized that UCHL1 deficiency attenuates PAH development by means of reductions in AKT1. METHODS: Tissues from animal pulmonary hypertension models as well as human pulmonary artery endothelial cells from patients with PAH exhibited increased vascular UCHL1 staining and protein expression. Exposure to LDN57444, a UCHL1-specific inhibitor, reduced human pulmonary artery endothelial cell and smooth muscle cell proliferation. Across 3 preclinical PAH models, LDN57444-exposed animals, Uchl1 knockout rats (Uchl1-/-), and conditional Uchl1 knockout mice (Tie2Cre-Uchl1fl/fl) demonstrated reduced right ventricular hypertrophy, right ventricular systolic pressures, and obliterative vascular remodeling. Lungs and pulmonary artery endothelial cells isolated from Uchl1-/- animals exhibited reduced total and activated Akt with increased ubiquitinated Akt levels. UCHL1-silenced human pulmonary artery endothelial cells displayed reduced lysine(K)63-linked and increased K48-linked AKT1 levels. RESULTS: Supporting experimental data, we found that rs9321, a variant in a GC-enriched region of the UCHL1 gene, is associated with reduced methylation (n=5133), increased UCHL1 gene expression in lungs (n=815), and reduced cardiac index in patients (n=796). In addition, Gadd45α (an established demethylating gene) knockout mice (Gadd45α-/-) exhibited reduced lung vascular UCHL1 and AKT1 expression along with attenuated hypoxic pulmonary hypertension. CONCLUSIONS: Our findings suggest that UCHL1 deficiency results in PAH attenuation by means of reduced AKT1, highlighting a novel therapeutic pathway in PAH.

Managing Pulmonary Arterial Hypertension With Cardiopulmonary Comorbidities.

Pulmonary arterial hypertension (PAH) and pulmonary hypertension associated with left-sided heart and lung diseases are most commonly easily discriminated and treated accordingly. With the changing epidemiology of PAH, however, a growing proportion of patients at the time of diagnosis present with comorbidities of varying severity. In addition to classical PAH, two distinct phenotypes have emerged: a heart failure with preserved ejection fraction-like phenotype and a lung phenotype. Importantly, the evidence supporting the currently proposed treatment algorithm for PAH has been generated mainly from PAH trials in which patients with cardiopulmonary comorbidities have been underrepresented or excluded. As a consequence, the best therapeutic approach for patients with common PAH with cardiopulmonary comorbidities remains largely unknown and requires further investigation. The present article reviews the relevant literature on the topic and describes the authors' views on the current therapeutic approach for these patients.

Single-cell and Spatial Transcriptomics Identified Fatty Acid-binding Proteins Controlling Endothelial Glycolytic and Arterial Programming in Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is a devastating disease characterized by obliterative vascular remodeling and persistent increase of vascular resistance, leading to right heart failure and premature death. Understanding the cellular and molecular mechanisms will help develop novel therapeutic approaches for PAH patients. Single-cell RNA sequencing (scRNAseq) analysis found that both FABP4 and FABP5 were highly induced in endothelial cells (ECs) of Egln1Tie2Cre (CKO) mice, which was also observed in pulmonary arterial ECs (PAECs) from idiopathic PAH (IPAH) patients, and in whole lungs of pulmonary hypertension (PH) rats. Plasma levels of FABP4/5 were upregulated in IPAH patients and directly correlated with severity of hemodynamics and biochemical parameters using plasma proteome analysis. Genetic deletion of both Fabp4 and 5 in CKO mice (Egln1Tie2Cre/Fabp4-5-/- ,TKO) caused a reduction of right ventricular systolic pressure (RVSP) and RV hypertrophy, attenuated pulmonary vascular remodeling and prevented the right heart failure assessed by echocardiography, hemodynamic and histological analysis. Employing bulk RNA-seq and scRNA-seq, and spatial transcriptomic analysis, we showed that Fabp4/5 deletion also inhibited EC glycolysis and distal arterial programming, reduced ROS and HIF-2α expression in PH lungs. Thus, PH causes aberrant expression of FABP4/5 in pulmonary ECs which leads to enhanced ECs glycolysis and distal arterial programming, contributing to the accumulation of arterial ECs and vascular remodeling and exacerbating the disease.

Hypusine Signaling Promotes Pulmonary Vascular Remodeling in Pulmonary Arterial Hypertension.

RATIONALE: The ubiquitous polyamine spermidine is essential for cell survival and proliferation. One important function of spermidine is to serve as a substrate for hypusination, a post-translational modification process that occurs exclusively on eukaryotic translation factor 5A (eIF5A) and ensures efficient translation of various gene products. Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by progressive obliteration of the small pulmonary arteries (PAs) due to excessive proliferation of PA smooth muscle cells (PASMCs) and suppressed apoptosis. OBJECTIVES: To characterize the role of hypusine signaling in PAH. METHODS: Molecular, genetic, and pharmacological approaches were used both in vitro and in vivo to investigate the role of hypusine signaling in pulmonary vascular remodeling. MEASUREMENTS AND MAIN RESULTS: Hypusine forming enzymes (deoxyhypusine synthase, DHPS and deoxyhypusine hydroxylase, DOHH) and hypusinated eIF5A are overexpressed in distal PAs and isolated PASMCs from PAH patients and animal models. In vitro, inhibition of DHPS using GC7 or short hairpin RNA resulted in a decrease in PAH-PASMC resistance to apoptosis and proliferation. In vivo, inactivation of one allele of Dhps targeted to smooth muscle cells alleviates PAH in mice and that its pharmacological inhibition significantly decreases pulmonary vascular remodeling and improves hemodynamics and cardiac function in two rat models of established PAH. Using mass spectrometry, we show that hypusine signaling promotes the expression of a broad array of proteins involved in oxidative phosphorylation, thus supporting the bioenergetic requirements of cell survival and proliferation. CONCLUSIONS: These findings support inhibiting hypusine signaling as a potential treatment for PAH.

Elastogenic potential and antisagging properties of a novel Murraya koenigii extract.

BACKGROUND: The process by which functional elastic fibers are produced, namely elastogenesis, is complex and difficult to assess in vitro. Identifying efficient elasticity-boosting ingredients thus represents a challenge. AIMS: The elasticity-boosting properties of a novel extract of Murraya koenigii leafy stems were assessed in vitro in 3D culture models before being evaluated in human female volunteers. METHODS: Synthesis of elastic fiber related proteins was evaluated in a skin-equivalent model. Using multiphoton microscopy, the structural organization of elastin deposits was studied within a scaffold-free dermal microtissue. Biomechanical properties of the 3D microtissue were also measured by atomic force microscopy. In vivo, fringe-projection and image analysis were used to evaluate nasogenian fold severity in a panel of Caucasian female volunteers. The impact of gravity on visible signs of facial aging was assessed by clinical scoring carried out alternatively in the supine and sitting positions. RESULTS: We showed the Murraya koenigii extract increased protein expressions of elastin and fibrillin-1 in a 3D skin equivalent model. Using scaffold-free dermal microtissue, we confirmed that Murraya koenigii extract allowed a proper and ordered network of elastin deposits and consequently improved tissue elasticity. Clinical data showed that a twice-daily application for 98 days of the extract formulated at 1% allowed to visibly reduce nasogenian fold severity, jowl severity and to mitigate the impact of gravity on the facial signs of aging. CONCLUSION: The newly discovered extract of Murraya koenigii leafy stems represents an innovative antiaging ingredient suited for elasticity-boosting and antisagging claims.

Right Ventricle and Epigenetics: A Systematic Review.

There is an increasing recognition of the crucial role of the right ventricle (RV) in determining the functional status and prognosis in multiple conditions. In the past decade, the epigenetic regulation (DNA methylation, histone modification, and non-coding RNAs) of gene expression has been raised as a critical determinant of RV development, RV physiological function, and RV pathological dysfunction. We thus aimed to perform an up-to-date review of the literature, gathering knowledge on the epigenetic modifications associated with RV function/dysfunction. Therefore, we conducted a systematic review of studies assessing the contribution of epigenetic modifications to RV development and/or the progression of RV dysfunction regardless of the causal pathology. English literature published on PubMed, between the inception of the study and 1 January 2023, was evaluated. Two authors independently evaluated whether studies met eligibility criteria before study results were extracted. Amongst the 817 studies screened, 109 studies were included in this review, including 69 that used human samples (e.g., RV myocardium, blood). While 37 proposed an epigenetic-based therapeutic intervention to improve RV function, none involved a clinical trial and 70 are descriptive. Surprisingly, we observed a substantial discrepancy between studies investigating the expression (up or down) and/or the contribution of the same epigenetic modifications on RV function or development. This exhaustive review of the literature summarizes the relevant epigenetic studies focusing on RV in human or preclinical setting.

Patients' perceptions on clinical trials outcomes in pulmonary arterial hypertension therapy.

The value placed by patients and their caregivers on the components of composite outcomes in pulmonary arterial hypertension (PAH) remains unknown. We surveyed the importance of these outcomes from a patients' and caregivers' perspective, with participants (n=335, including 257 patients with PAH) rating individual components defining clinical worsening in PAH trials as of critical, major, mild-to-moderate or minor importance. Most outcomes were considered of major or mild-to-moderate importance to patients. Death was the only outcome considered of critical importance. Perceptions of clinical outcomes varied between patients and caregivers. Integrating patients' perception in the elaboration of clinical trials is essential.

The future of group 2 pulmonary hypertension: Exploring clinical trials and therapeutic targets.

Pulmonary hypertension due to left heart disease (PH-LHD) or group 2 PH is the most common and lethal form of PH, occurring secondary to left ventricular systolic or diastolic heart failure (HF), left-sided valvular diseases, and congenital abnormalities. It is subdivided into isolated postcapillary PH (IpcPH) and combined pre- and post-capillary PH (CpcPH), with the latter sharing many similarities with group 1 PH. CpcPH is associated with worse outcomes and increased morbidity and mortality when compared to IpcPH. Although IpcPH can be improved by treatment of the underlying LHD, CpcPH is an incurable disease for which no specific treatment exists, likely due to the lack of understanding of its underlying mechanisms. Furthermore, drugs approved for PAH are not recommended for group 2 PH, as they are either ineffective or even deleterious. With this major unmet medical need, a better understanding of mechanisms and the identification of effective treatment strategies for this deadly condition are urgently needed. This review presents relevant background of the molecular mechanisms underlying PH-LHD that could translate into innovative therapeutic targets and explores novel targets currently being evaluated in clinical trials.

Targeting Wnt-ß-Catenin-FOSL Signaling Ameliorates Right Ventricular Remodeling.

BACKGROUND: The ability of the right ventricle (RV) to adapt to an increased pressure afterload determines survival in patients with pulmonary arterial hypertension. At present, there are no specific treatments available to prevent RV failure, except for heart/lung transplantation. The wingless/int-1 (Wnt) signaling pathway plays an important role in the development of the RV and may also be implicated in adult cardiac remodeling. METHODS: Molecular, biochemical, and pharmacological approaches were used both in vitro and in vivo to investigate the role of Wnt signaling in RV remodeling. RESULTS: Wnt/ß-catenin signaling molecules are upregulated in RV of patients with pulmonary arterial hypertension and animal models of RV overload (pulmonary artery banding-induced and monocrotaline rat models). Activation of Wnt/ß-catenin signaling leads to RV remodeling via transcriptional activation of FOSL1 and FOSL2 (FOS proto-oncogene [FOS] like 1/2, AP-1 [activator protein 1] transcription factor subunit). Immunohistochemical analysis of pulmonary artery banding -exposed BAT-Gal (ß-catenin-activated transgene driving expression of nuclear ß-galactosidase) reporter mice RVs exhibited an increase in ß-catenin expression compared with their respective controls. Genetic inhibition of ß-catenin, FOSL1/2, or WNT3A stimulation of RV fibroblasts significantly reduced collagen synthesis and other remodeling genes. Importantly, pharmacological inhibition of Wnt signaling using inhibitor of PORCN (porcupine O-acyltransferase), LGKK-974 attenuated fibrosis and cardiac hypertrophy leading to improvement in RV function in both, pulmonary artery banding - and monocrotaline-induced RV overload. CONCLUSIONS: Wnt- ß-Catenin-FOSL signaling is centrally involved in the hypertrophic RV response to increased afterload, offering novel targets for therapeutic interference with RV failure in pulmonary hypertension.

G9a/GLP Targeting Ameliorates Pulmonary Vascular Remodeling in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is characterized by progressive vascular remodeling of small pulmonary arteries (PAs) causing sustained elevation of PA pressure, right ventricular failure, and death. Similar to cancer cells, PA smooth muscle cells (PASMCs), which play a key role in pulmonary vascular remodeling, have adopted multiple mechanisms to sustain their survival and proliferation in the presence of stress. The histone methyltransferase G9a and its partner protein GLP (G9a-like protein) have been shown to exert oncogenic effects and to serve as a buffer against an exaggerated transcriptional response. Therefore, we hypothesized that upregulation of G9a and GLP in PAH plays a pivotal role in pulmonary vascular remodeling by maintaining the abnormal phenotype of PAH-PASMCs. We found that G9a is increased in PASMCs from patients with PAH as well as in remodeled PAs from animal models. Pharmacological inhibition of G9a/GLP activity using BIX01294 and UNC0642 significantly reduced the prosurvival and proproliferative potentials of cultured PAH-PASMCs. Using RNA sequencing, further exploration revealed that G9a/GLP promotes extracellular matrix production and affords protection against the negative effects of an overactive stress response. Finally, we found that therapeutic treatment with BIX01294 reduced pulmonary vascular remodeling and lowered mean PA pressure in fawn-hooded rats. Treatment of Sugen/hypoxia-challenged mice with BIX01294 also improved pulmonary hemodynamics and right ventricular function. In conclusion, these findings indicate that G9a/GLP inhibition may represent a new therapeutic approach in PAH.

Right Ventricular Maladaptation to Pressure Overload in Fischer Rats Is Associated With Profound Deficiency in Adenylate Kinase 1 and Impaired Ventricular Energetics.

BACKGROUND: We explored the mechanism of maladaptive right ventricular (RV) remodeling in Fischer compared with Sprague-Dawley (SD) rats exposed to pressure overload. METHODS: Pulmonary hypertension was induced by injection of the VEGFR antagonist, SU5416, followed by a 3-week exposure to hypoxia (Sugen chronic hypoxia). In vivo oxidative metabolism was assessed by RV/left ventricle ratio of [11C]acetate positron emission tomography clearance (kmono). Unbiased, global transcriptional and proteomic profiling was performed in Fischer and SD rats at baseline and after Sugen chronic hypoxia. RESULTS: All Fischer rats succumbed to RV failure by 5 weeks, whereas SD rats showed preserved RV function and 88% survival beyond 9 weeks (P<0.0001). Fischer rats exhibited increased oxidative metabolism at 4 weeks (P<0.05) and impaired RV efficiency compared with SD (work metabolic index: 52±10 versus 91±27 mmHg·mL/cm2, respectively; P<0.05), but no differences in mitochondrial complex activity. AK1 (adenylate kinase 1) was among the top 10 differentially expressed genes between Fischer and SD rats, with markedly lower RV expression in Fischer rats (FC: 3.36, P<0.05), confirmed by proteomic analysis and validated by Western blotting (>10-fold reduction, P<0.001). While whole-genome sequencing failed to reveal any coding region mutations in Fischer rats, there was a unique variant in a highly conserved upstream flanking region likely involved in the regulation of AK1 expression. CONCLUSIONS: Therefore, Fischer rats exhibit profound AK1 deficiency and inefficient cardiac energetics likely related to reduced adenosine triphosphate shuttling from the mitochondria to the contractile fibers. This represents a novel mechanism for RV failure in response to chronic increases in afterload.

Intra-Airway Gene Delivery for Pulmonary Hypertension in Rodent Models.

Pulmonary arterial hypertension (PAH) is a severe and progressive cardiopulmonary disease characterized by pathological remodeling of the resistance pulmonary arteries (PA), ultimately leading to right ventricular (RV) failure and death. Animal models have been particularly useful for unraveling the pathogenesis of PAH by providing incisive experimental strategies that were impossible in human studies. Over the past decade, gene therapy has been making considerable progress as an alternative strategy for treating PAH disease. Animal models mimicking PAH disease are essential at preclinical stages for assessing the therapeutic potential of gene therapy and determining genome viral vectors transduction, safety, dosage, and localization of transgene expression. The most commonly used PAH rat models in gene therapy studies are the monocrotaline (MCT), the chronic hypoxia-Sugen 5416, and the pneumonectomy (PNT)-MCT models. Here, we provide detailed protocols for creating these preclinical rodent models of PAH commonly used to assess the efficiency of lung gene therapy in PAH.

Assessment of Clinical Worsening End Points as a Surrogate for Mortality in Pulmonary Arterial Hypertension: A Systematic Review and Meta-Analysis of Randomized Controlled Trials.

BACKGROUND: Clinical worsening (CW) is a composite end point commonly used in pulmonary arterial hypertension (PAH) trials. We aimed to assess the trial-level surrogacy of CW for mortality in PAH trials, and whether the various CW components were similar in terms of frequency of occurrence, treatment-related relative risk (RR) reduction, and importance to patients. METHODS: We searched MEDLINE, Embase, and the Cochrane Library (January 1990 to December 2020) for trials evaluating the effects of PAH therapies on CW. The coefficient of determination between the RR for CW and mortality was assessed by regression analysis. The frequency of occurrence, RR reduction, and importance to patients of the CW components were assessed. RESULTS: We included 35 independent cohorts (9450 patients). PAH therapies significantly reduced CW events (RR, 0.64 [95% CI, 0.55-0.73]), including PAH-related hospitalizations (RR, 0.61 [95% CI, 0.47-0.79]), treatment escalation (RR, 0.57 [95% CI, 0.38-0.84]) and symptomatic progression (RR, 0.58 [95% CI, 0.48-0.69]), and modestly reduced all-cause mortality when incorporating deaths occurring after a primary CW-defining event (RR, 0.860 [95% CI, 0.742-0.997]). However, the effects of PAH-specific therapies on CW only modestly correlated with their effects on mortality (R2trial, 0.35 [95% CI, 0.10-0.59]; P<0.0001), and the gradient in the treatment effect across component end points was large in the majority of trials. The weighted proportions of CW-defining events were hospitalization (33.5%) and symptomatic progression (32.3%), whereas death (6.7%), treatment escalation (5.6%), and transplantation/atrioseptostomy (0.2%) were infrequent. CW events were driven by the occurrence of events of major (49%) and mild-to-moderate (37%) importance to patients, with 14% of the events valued as critical. CONCLUSIONS: PAH therapies significantly reduced CW events, but study-level CW is not a surrogate for mortality in PAH trials. Moreover, components of CW largely vary in frequency, response to therapy, and importance to patients and are thus not interchangeable. REGISTRATION: URL: https://www.crd.york.ac.uk/PROSPERO; Unique identifier: CRD42020178949.

Epigenetic reactivation of transcriptional programs orchestrating fetal lung development in human pulmonary hypertension.

Phenotypic alterations in resident vascular cells contribute to the vascular remodeling process in diseases such as pulmonary (arterial) hypertension [P(A)H]. How the molecular interplay between transcriptional coactivators, transcription factors (TFs), and chromatin state alterations facilitate the maintenance of persistently activated cellular phenotypes that consequently aggravate vascular remodeling processes in PAH remains poorly explored. RNA sequencing (RNA-seq) in pulmonary artery fibroblasts (FBs) from adult human PAH and control lungs revealed 2460 differentially transcribed genes. Chromatin immunoprecipitation sequencing (ChIP-seq) revealed extensive differential distribution of transcriptionally accessible chromatin signatures, with 4152 active enhancers altered in PAH-FBs. Integrative analysis of RNA-seq and ChIP-seq data revealed that the transcriptional signatures for lung morphogenesis were epigenetically derepressed in PAH-FBs, including coexpression of T-box TF 4 (TBX4), TBX5, and SRY-box TF 9 (SOX9), which are involved in the early stages of lung development. These TFs were expressed in mouse fetuses and then repressed postnatally but were maintained in persistent PH of the newborn and reexpressed in adult PAH. Silencing of TBX4, TBX5, SOX9, or E1A-associated protein P300 (EP300) by RNA interference or small-molecule compounds regressed PAH phenotypes and mesenchymal signatures in arterial FBs and smooth muscle cells. Pharmacological inhibition of the P300/CREB-binding protein complex reduced the remodeling of distal pulmonary vessels, improved hemodynamics, and reversed established PAH in three rodent models in vivo, as well as reduced vascular remodeling in precision-cut tissue slices from human PAH lungs ex vivo. Epigenetic reactivation of TFs associated with lung development therefore underlies PAH pathogenesis, offering therapeutic opportunities.