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Cardiomyocyte GC1 Mediates Estrogenic Angiogenesis in Right Heart Remodeling.
No full textBACKGROUND
Right ventricular (RV) dysfunction increases mortality in heart failure and pulmonary hypertension. However, women demonstrate better RV function and survival than men. This difference is attributed to estrogen, though mechanistic details remain unclear. Given estrogen's stimulation of NO production, we investigated whether and how cardiomyocyte NO-sensitive soluble GC1 (guanylyl cyclase) mediates female-specific, adaptive RV pressure-overload remodeling.
METHODS
Adult male and female mice with cardiomyocyte-specific GC1 deficiency (cardiomyocyte-specific knockout) and littermate controls underwent pulmonary artery banding (PAB) or thoracotomy (Sham). At 6-week postsurgery, RV function was assessed via echocardiography, pressure-volume loops, and treadmill testing. RV function, histopathology, and transcript profiles were compared across sex, genotype, and surgical group. Single-nucleus RNA sequencing of RV tissue was performed to identify putative cardiomyocyte GC1-mediated cell-cell communication in adaptive RV pressure-overload remodeling. Endothelial coculture assays with controls versus cardiomyocyte-specific knockout cardiomyocytes evaluated estrogen and cardiomyocyte GC1-dependence of the identified intercellular signaling.
RESULTS
Female controls PAB adapted RV contractility to overcome RV pressure-overload, thereby preserving RV-PA coupling. In contrast, female cardiomyocyte-specific knockout, ovariectomized female controls, and male PAB developed severe RV dysfunction with RV-PA uncoupling. These groups with maladapted RVs had marked cardiomyocyte hypertrophy, interstitial fibrosis, and capillary rarefaction; female control PAB had minimal changes. Among histological features, the capillary-to-cardiomyocyte ratio showed the strongest correlation with RV function. Ratios were similar between female control PAB and Sham, but abnormally low in all other PAB. Single-nucleus RNA sequence and coculture analyses revealed that cardiomyocyte GC1 is central to Vegf (vascular endothelial growth factor)-Vegfr proangiogenic signaling from cardiomyocytes to endothelial cells in the adaptively remodeled, pressure-overloaded RV.
CONCLUSIONS
We identified a novel estrogen- and cardiomyocyte GC1-dependent pathway that mitigates capillary rarefaction, maintaining normal capillary-to-cardiomyocyte ratio and preserving RV-PA coupling under RV pressure-overload. This proangiogenic, estrogen- and cardiomyocyte GC1-dependent mechanism contributes to sex-specific differences in RV remodeling and may inform the development of targeted therapies for RV dysfunction
Engineered 3D kidney glomerular microtissues to model podocyte-centric diseases for the validation of new drug targets
No full textPodocytopathies are a class of kidney diseases characterized by podocyte injury, driving proteinuria, and reduced kidney function. Podocyte injury often disrupts cytoskeletal dynamics and cellular adhesion leading to glomerular dysfunction. The lack of in vitro models truly recapitulating the three-dimensional (3D) organization and tissue mechanics, limits the understanding of podocyte pathophysiology and therapeutic development. Here, we developed 3D microtissues that recapitulate the structure and mechanics of the glomerular filtration barrier, enabling modeling of chemically and genetically induced podocyte injuries for potential new drug target validation. These microtissues replicate the three-layer glomerular structure and hemodynamic-like mechanical stretch, providing an in vitro platform to evaluate disease-relevant mechanobiological signaling, podocyte cytoskeletal dynamics, and adhesion to the basement membrane. We measured collective cellular forces to assess podocyte biochemical resilience in response to genetic or chemical injuries. As a proof-of-concept, we modeled podocyte injury through overexpressing TRPC6 (Transient Receptor Potential Canonical 6), a genetically validated target in podocytopathies, and assessed the therapeutic potential of the inhibitor SAR7334. Our results demonstrated a loss of podocyte contractile forces upon TRPC6 overexpression and recovery following inhibitor treatment, highlighting the potential of glomerular microtissues to model podocyte mechano-pathophysiology and serve as a robust platform for screening new therapies
Defining Non-small Cell Lung Cancer Tumor Microenvironment Changes at Primary and Acquired Immune Checkpoint Inhibitor Resistance Using Clinical and Real-World Data.
No full textImmune checkpoint inhibitors (ICI) have demonstrated clinical efficacy in non-small cell lung cancer (NSCLC), and extensive research has been conducted to explore biomarkers predictive of ICI response. However, the impact of ICI on the tumor and tumor microenvironment at primary and acquired resistance states is understudied due to the difficulty of collecting tissue biopsies at disease progression. In this study, we leveraged clinical and real-world data to study ICI resistance. Data used in this work consist of treatment outcome information and tissue RNA sequencing data from advanced-stage NSCLC cohorts from three sources: the Tempus real-world evidence database; CANOPY-1 (NCT03631199), a phase III clinical trial in first-line NSCLC; and Stand Up To Cancer (SU2C) publication. Our results indicate higher IFNγ and T-cell exhaustion in patients' tumors at acquired resistance and low levels of B-cell and dendritic cell expression at primary resistance. The lower B-cell and dendritic cell levels may be primarily driven by prior treatment with a platinum-based chemotherapy regimen. Baseline transcriptomics data additionally suggest that innate immune cells may play an antitumor role in PD-L1<1% patients, whereas IFNγ and T-cell inflammation are more predictive of ICI treatment outcomes in PD-L1≥1% patients. Our study suggests a clear divergence of the tumor microenvironment in patients with primary versus acquired resistance and a potential role of myeloid cells in the PD-L1<1% population. These findings shed light on potential next-generation therapies to overcome ICI resistance.ICI benefits patients with NSCLC, but resistance remains common. Our research highlights differences in tumor environments between primary and acquired resistance after ICI treatment, emphasizing distinct post-therapy approaches. Findings also suggest myeloid cells as key players in PD-L1-negative cases, guiding future treatment strategies to overcome resistance and improve outcomes
An industry perspective on clinical development and regulatory strategies for subcutaneously administered high-dose biologics.
No full textThe Subcutaneous Drug Development and Delivery Consortium aims to advance the technical and regulatory understanding that can enable development of subcutaneous biotherapeutics towards improved outcomes. The Consortium's Clinical Development and Regulatory Strategy sub team conducted a cross-company survey to explore strategies, challenges, and best practices in developing subcutaneous products for high-dose biologics. With the growing prevalence of biologics, subcutaneous delivery is increasingly recognized for its potential to reduce healthcare burdens and enhance treatment convenience in treatment administered by healthcare providers, caregivers or individual users. The survey results indicate that industry participants view high-dose subcutaneous formulations as beneficial for customer-centered care, reducing clinical visits and enabling at-home administration. The primary drivers for subcutaneous development include improved experience with the parenteral dosing regimen, increased adherence, and continuous improvement towards emerging needs (lifecycle management). While companies are increasingly leveraging enabling technologies such as permeation enhancers, high concentration technologies, and wearable devices to resolve technical barriers with delivering high doses subcutaneously, challenges remain. These challenges can range from user acceptance to regulatory complexities with implementation of innovation and clinical strategies. One of the key opportunities identified is the advancement of bridging strategies to transition from intravenous to subcutaneous formulation, informed by pharmacokinetic/ pharmacodynamic modeling and simulation and pharmacokinetic clinical bridging studies. The survey highlights the need for continued collaboration and innovation to optimize subcutaneous high-dose biologic development, aligning industry practices with evolving regulatory and customer-centric demands
Teratogenic Risk Impact Mitigation (TRIM): Development of Explicit Criteria to Facilitate Decisions Regarding Teratogenic Risk Mitigation Strategies.
No full textPreventing fetal exposure to teratogenic medications is an important target for risk mitigation efforts. Decisions about risk mitigation efforts specific to teratogenic medications are complex.The Teratogenic Risk Impact and Mitigation (TRIM) tool was developed as an innovative decision support tool to facilitate prioritization of teratogenic medications for risk mitigation strategies.We employed a modified Delphi study design involving experts across teratology, obstetrics/gynecology, and medication safety. Panelists proposed decision criteria in three focus groups, followed by e-Delphi rounds to reach a consensus on criteria regarding three dimensions: (1) completeness; (2) relevance; and (3) distinctiveness. Aggregated feedback from each round was used to inform revision of the criteria in subsequent rounds.A total of 33 candidate criteria proposed by 32 focus group participants were organized into ten distinct criteria for the Delphi process. Consensus (defined as > 85% agreement on all three dimensions) was reached after three e-Delphi rounds, resulting in six criteria: (1) background use among persons of reproductive potential; (2) overall medication benefit considering severity of the indication and availability of alternatives; (3) seriousness of the teratogenic outcome; (4) risk of the teratogenic outcome; (5) certainty regarding teratogenicity; and (6) the risk of exposure during pregnancy.We established measurable criteria to inform decisions when prioritizing teratogenic medications for risk mitigation programs. Criteria are consensus based and consistent with relevant regulatory guidance. Future work will operationalize these criteria and determine specific weights to facilitate medication-specific TRIM scores. Through its explicit framework, the TRIM tool may support consistent, transparent, and rational decision making and help optimize the contribution of risk mitigation programs to public health
Structural changes of cinnarizine-stabilizer core-shell nano- and micro-suspensions following freeze- and spray-drying determined from dynamic nuclear polarization enhanced NMR.
No full textDeveloping potent drug molecules that are also highly soluble in aqueous media puts strong constraints on molecular design. As a result, there is intense interest today in developing drug formulation strategies that increase solubility. Specifically, nanosizing involves the reduction of the particle size of the active pharmaceutical ingredient (API) to the sub-micron range, which increases the surface area and dissolution rate. This strategy requires the addition of stabilizers, generally selected through extensive experimental screening, to maintain the desired physical properties over time. To better understand stabilization mechanisms and to develop better future formulations, atomic-level characterization of the particle structures is required in terms of both the size and spatial distribution of the components and the interactions. However, methods that can simultaneously provide this information are scarce. Here, using cinnarizine as a model for nanosuspensions, we show that by using DNP-enhanced NMR we can (i) detect and assign the API and the stabilizers present in formulations; (ii) observe atomic-level API-stabilizer interactions at natural isotopic abundance using two-dimensional 1H-13C correlation NMR experiments; and (iii) determine the domain sizes and the hierarchical structure of the API-stabilizer particles on the nano-meter length scale, based on polarization build-up curves and steady-state enhancements. We then use this approach to evaluate how freeze-drying and spray drying processes, generally used to isolate the material in the solid state, impact the particle structure. More broadly, the results confirm the applicability of DNP-enhanced NMR methods to characterize pharmaceutical suspensions or slurries, and to follow changes upon further processing
Innovative Medicines Initiative public-private partnerships to enhance translational safety.
No full textInnovative Medicines Initiative projects focused on translational safety have developed a range of tools to improve preclinical assessments of potential drug toxicities, and demonstrated the value of integrating data from multiple companies
Modulating pro-fibrotic macrophages using yeast beta-glucan microparticles prepared by Pressurized Gas eXpanded liquid (PGX) Technology®
No full textPro-fibrotic M2-like macrophages are widely implicated in the pathogenesis and progression of lung fibrosis due to their production of pro-fibrotic growth factors and cytokines. Yeast beta-glucan (YBG) microparticles have shown potential as immunomodulators that can convert macrophage polarization from a pro-fibrotic phenotype to an anti-fibrotic phenotype through the engagement of the Dectin-1 receptor. However, the processing conditions used to fabricate YBG microparticles can lead to unpredictable immunomodulatory effects. Herein, we report the use of Pressurized Gas eXpanded liquids (PGX) Technology® to fabricate YBG (PGX-YBG) microparticles with higher surface areas, lower densities, and smaller and more uniform size distributions compared to commercially available spray-dried YBGs. PGX-YBG is shown to activate Dectin-1 more efficiently in vitro while avoiding significant TLR 2/4 activation. Furthermore, PGX-YBG microparticles effectively modulate M2-like fibrosis-inducing murine and human macrophages into fibrosis-suppressing macrophages both in vitro as well as in ex vivo precision-cut murine lung slices, suggesting their potential utility as a therapeutic for addressing a broad spectrum of fibrotic end-point lung diseases
Single cell multi-omics reveals re-dosing with CD3 bispecific antibody induces a TCF7 high central memory CD8 + T cell population associated with reduced cytokine production.
No full textT cell engaging therapies are commonly accompanied by excessive cytokine production and risk of cytokine release syndrome (CRS). Intriguingly, CRS risk with CD3-engaging bispecific antibody (BSP) is primarily limited to the first dose, termed the first-dose effect. Mechanisms underlying this effect remain unknown. CD3 bispecific induces cytokine cascade via T cell triggering and bystander cells. We hypothesize that distinct T cell biology between doses drives the first-dose effect.We used the Re-directed T Cell Cytotoxicity (RTCC) assay to assess tumor killing and cytokine production by human donor T cells after initial versus subsequent CD3/CD20 BSP treatment. After confirming the first-dose effect in the experimental system containing only T cells and target tumor cells, we employed 10x Genomics single cell multi-omics to study the molecular mechanisms.Compared with initial CD3/CD20 BSP treatment, subsequent treatment exhibited lower cytokine levels and comparable tumor killing. Single cell multi-omics unveiled distinct T cell biology. In initial treatment, T effector memory (Tem) cells are the primary cells that respond to CD3 bispecific antibody stimulus by producing moderate levels of cytolytic and high levels of cytokine gene transcription. In the subsequent treatment, a new population of high TCF7 expressing central memory CD8 + cells (CD8-Tcm-TCF7), possibly originated from stimulated naive T cells, are the primary responding cells that produce a shifted balance with high level of the cytolytic gene transcription (GZMB) and low level of cytokine gene transcription (TNF-alpha and IFN-gamma). Dasatinib co-treatment during initial treatment eliminated cytolytic activity and cytokine production, allowing uncompromised tumor killing and reduced cytokine production upon re-challenge.The distinct T cell populations that respond to first and subsequent CD3 bispecific treatment offer an explanation to the first-dose effect, wherein the risk of CRS associated with CD3 bispecific treatment is mainly limited to the initial dose. Furthermore, our work suggests that tumor killing capacity and cytokine production of T cells could be uncoupled, as demonstrated here by utilizing different T cell populations as effector cells. These findings could be further explored for designing mechanism-based strategies to mitigate the risk of CRS
Characterizing Antisense Oligonucleotide-Induced Histopathology Findings in Spinal Cord of Mauritius Cynomolgus Monkeys by Molecular Localization Investigation.
No full textThe safety of a 2'-O-methoxyethyl antisense oligonucleotide (ASO) was investigated in Mauritius cynomolgus monkeys in a 41-week Good Laboratory Practice (GLP) toxicity study after multiple intrathecal (IT) administrations. Histopathological examination revealed ectopic formation of lymphoid follicles in the spinal cord (SC) at the injection site at all doses and the presence of granular material in neurons of the SC in high-dose animals. The granular material was seen in all the segments of the SC, but mainly in the lumbar segment and persisted at the end of the 26-week recovery period, while the lymphoid follicles showed a reversibility trend. Findings associated with repeated IT administration of ASOs have been described in nonhuman primate (NHP) toxicity studies, specifically in the brain, but findings in the SC are rarely reported. In the present study, we report a high incidence of findings in the SC compared to brain, especially in the lumbar segment in proximity to IT injection sites. An extensive panel of immunohistochemistry markers showed that the ectopic lymphoid follicle formation (LFF) had a cellular composition and organization consistent with tertiary lymphoid structure (TLS) without associated axonal damage in the adjacent nervous tissue. In situ hybridization with an miRNA probe complementary to the ASO revealed that the granular material represented a dose-dependent ASO accumulation in the cytoplasm of neurons without inducing cell death or apoptosis. Glial and ependymal cells in the SC also showed dose-dependent accumulation of the ASO preceding detection of granular material by hematoxylin and eosin (H&E). Based on these molecular localization data, the presence of LFF in SC suggests a chronic local immune activation. Considering the absence of neuronal dysfunction or injury and transient clinical signs previously reported with other 2'-MOE ASOs, the presence of TLS and ASO was considered non-adverse