Modeling congenital heart disease: lessons from mice, hPSC-based models, and organoids.

Congenital heart defects (CHDs) are among the most common birth defects, but their etiology has long been mysterious. In recent decades, the development of a variety of experimental models has led to a greater understanding of the molecular basis of CHDs. In this review, we contrast mouse models of CHD, which maintain the anatomical arrangement of the heart, and human cellular models of CHD, which are more likely to capture human-specific biology but lack anatomical structure. We also discuss the recent development of cardiac organoids, which are a promising step toward more anatomically informative human models of CHD.

Brahma safeguards canalization of cardiac mesoderm differentiation.

Differentiation proceeds along a continuum of increasingly fate-restricted intermediates, referred to as canalization1,2. Canalization is essential for stabilizing cell fate, but the mechanisms that underlie robust canalization are unclear. Here we show that the BRG1/BRM-associated factor (BAF) chromatin-remodelling complex ATPase gene Brm safeguards cell identity during directed cardiogenesis of mouse embryonic stem cells. Despite the establishment of a well-differentiated precardiac mesoderm, Brm-/- cells predominantly became neural precursors, violating germ layer assignment. Trajectory inference showed a sudden acquisition of a non-mesodermal identity in Brm-/- cells. Mechanistically, the loss of Brm prevented de novo accessibility of primed cardiac enhancers while increasing the expression of neurogenic factor POU3F1, preventing the binding of the neural suppressor REST and shifting the composition of BRG1 complexes. The identity switch caused by the Brm mutation was overcome by increasing BMP4 levels during mesoderm induction. Mathematical modelling supports these observations and demonstrates that Brm deletion affects cell fate trajectory by modifying saddle-node bifurcations2. In the mouse embryo, Brm deletion exacerbated mesoderm-deleted Brg1-mutant phenotypes, severely compromising cardiogenesis, and reveals an in vivo role for Brm. Our results show that Brm is a compensable safeguard of the fidelity of mesoderm chromatin states, and support a model in which developmental canalization is not a rigid irreversible path, but a highly plastic trajectory.

A disrupted compartment boundary underlies abnormal cardiac patterning and congenital heart defects.

Failure of septation of the interventricular septum (IVS) is the most common congenital heart defect (CHD), but mechanisms for patterning the IVS are largely unknown. We show that a Tbx5+/Mef2cAHF+ progenitor lineage forms a compartment boundary bisecting the IVS. This coordinated population originates at a first- and second heart field interface, subsequently forming a morphogenetic nexus. Ablation of Tbx5+/Mef2cAHF+ progenitors cause IVS disorganization, right ventricular hypoplasia and mixing of IVS lineages. Reduced dosage of the CHD transcription factor TBX5 disrupts boundary position and integrity, resulting in ventricular septation defects (VSDs) and patterning defects, including Slit2 and Ntn1 misexpression. Reducing NTN1 dosage partly rescues cardiac defects in Tbx5 mutant embryos. Loss of Slit2 or Ntn1 causes VSDs and perturbed septal lineage distributions. Thus, we identify essential cues that direct progenitors to pattern a compartment boundary for proper cardiac septation, revealing new mechanisms for cardiac birth defects.

Familial exudative vitreoretinopathy complicated with macular hole retinal detachment.

Familial exudative vitreoretinopathy (FEVR) is a rare genetically heterogeneous inherited disorder with varied presentation. We report a case of FEVR with a classic tractional component and a rare co-occurrence of macular hole with subtotal retinal detachment. The diagnosis was made on clinical examination and imaging. There have been four cases of full-thickness macular holes reported in the literature and none with macular hole retinal detachment. The patient underwent vitrectomy for the same and had silicone oil removal with attached retina postoperatively. We want to highlight this rare association and emphasize the importance of regular follow-up of FEVR cases which tend to progress after remaining quiescent for years.

Modeling Human TBX5 Haploinsufficiency Predicts Regulatory Networks for Congenital Heart Disease.

Haploinsufficiency of transcriptional regulators causes human congenital heart disease (CHD); however, the underlying CHD gene regulatory network (GRN) imbalances are unknown. Here, we define transcriptional consequences of reduced dosage of the CHD transcription factor, TBX5, in individual cells during cardiomyocyte differentiation from human induced pluripotent stem cells (iPSCs). We discovered highly sensitive dysregulation of TBX5-dependent pathways-including lineage decisions and genes associated with heart development, cardiomyocyte function, and CHD genetics-in discrete subpopulations of cardiomyocytes. Spatial transcriptomic mapping revealed chamber-restricted expression for many TBX5-sensitive transcripts. GRN analysis indicated that cardiac network stability, including vulnerable CHD-linked nodes, is sensitive to TBX5 dosage. A GRN-predicted genetic interaction between Tbx5 and Mef2c, manifesting as ventricular septation defects, was validated in mice. These results demonstrate exquisite and diverse sensitivity to TBX5 dosage in heterogeneous subsets of iPSC-derived cardiomyocytes and predicts candidate GRNs for human CHDs, with implications for quantitative transcriptional regulation in disease.

Synergy of nitric oxide and silver sulfadiazine against gram-negative, gram-positive, and antibiotic-resistant pathogens.

The synergistic activity between nitric oxide (NO) released from diazeniumdiolate-modified proline (PROLI/NO) and silver(I) sulfadiazine (AgSD) was evaluated against Escherichia coli, Enterococcus faecalis, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis using a modified broth microdilution technique and a checkerboard-type assay. The combination of NO and AgSD was defined as synergistic when the fractional bactericidal concentration (FBC) was calculated to be <0.5. Gram-negative species were generally more susceptible to the individual antimicrobial agents than the Gram-positive bacteria, while Gram-positive bacteria were more susceptible to combination therapy. The in vitro synergistic activity of AgSD and NO observed against a range of pathogens strongly supports future investigation of this therapeutic combination, particularly for its potential use in the treatment of burns and chronic wounds.

Degradable nitric oxide-releasing biomaterials via post-polymerization functionalization of cross-linked polyesters.

The synthesis of diverse nitric oxide (NO)-releasing network polyesters is described. The melt phase condensation of polyols with a calculated excess of diacid followed by thermal curing generates cross-linked polyesters containing acid end groups. Varying the composition and curing temperatures of the polyesters resulted in materials with tunable thermal and degradation properties. Glass transition temperatures for the synthesized materials range from -25.5 to 3.2 °C, while complete degradation of these polyesters occurs within a minimum of nine weeks under physiological conditions (pH 7.4, 37 °C). Post-polymerization coupling of aminothiols to terminal carboxylic acids generate thiol-containing polyesters, with thermal and degradation characteristics similar to those of the parent polyesters. After nitrosation, these materials are capable of releasing up to 0.81 µmol NO cm(-2) for up to 6 d. The utility of the polyesters as antibacterial biomaterials was indicated by an 80% reduction of Pseudomonas aeruginosa adhesion compared to unmodified controls.

Patronin-mediated minus end growth is required for dendritic microtubule polarity.

Microtubule minus ends are thought to be stable in cells. Surprisingly, in Drosophila and zebrafish neurons, we observed persistent minus end growth, with runs lasting over 10 min. In Drosophila, extended minus end growth depended on Patronin, and Patronin reduction disrupted dendritic minus-end-out polarity. In fly dendrites, microtubule nucleation sites localize at dendrite branch points. Therefore, we hypothesized minus end growth might be particularly important beyond branch points. Distal dendrites have mixed polarity, and reduction of Patronin lowered the number of minus-end-out microtubules. More strikingly, extra Patronin made terminal dendrites almost completely minus-end-out, indicating low Patronin normally limits minus-end-out microtubules. To determine whether minus end growth populated new dendrites with microtubules, we analyzed dendrite development and regeneration. Minus ends extended into growing dendrites in the presence of Patronin. In sum, our data suggest that Patronin facilitates sustained microtubule minus end growth, which is critical for populating dendrites with minus-end-out microtubules.

TAT-conjugated nanoparticles for the CNS delivery of anti-HIV drugs.

We have shown that nanoparticles (NPs) conjugated to trans-activating transcriptor (TAT) peptide bypass the efflux action of P-glycoprotein and increase the transport of the encapsulated ritonavir, a protease inhibitor (PI), across the blood-brain-barrier (BBB) to the central nervous system (CNS). A steady increase in the drug parenchyma/capillary ratio over time without disrupting the BBB integrity suggests that TAT-conjugated NPs are first immobilized in the brain vasculature prior to their transport into parenchyma. Localization of NPs in the brain parenchyma was further confirmed with histological analysis of the brain sections. The brain drug level with conjugated NPs was 800-fold higher than that with drug in solution at two weeks. Drug clearance was seen within four weeks. In conclusion, TAT-conjugated NPs enhanced the CNS bioavailability of the encapsulated PI and maintained therapeutic drug levels in the brain for a sustained period that could be effective in reducing the viral load in the CNS, which acts as a reservoir for the replicating HIV-1 virus.

Two Drosophila model neurons can regenerate axons from the stump or from a converted dendrite, with feedback between the two sites.

BACKGROUND: After axon severing, neurons recover function by reinitiating axon outgrowth. New outgrowth often originates from the remaining axon stump. However, in many mammalian neurons, new axons initiate from a dendritic site when the axon is injured close to the cell body. METHODS: Drosophila sensory neurons are ideal for studying neuronal injury responses because they can be injured reproducibly in a variety of genetic backgrounds. In Drosophila, it has been shown that a complex sensory neuron, ddaC, can regenerate an axon from a stump, and a simple sensory neuron, ddaE, can regenerate an axon from a dendrite. To provide a more complete picture of axon regeneration in these cell types, we performed additional injury types. RESULTS: We found that ddaE neurons can initiate regeneration from an axon stump when a stump remains. We also showed that ddaC neurons regenerate from the dendrite when the axon is severed close to the cell body. We next demonstrated if a stump remains, new axons can originate from this site and a dendrite at the same time. Because cutting the axon close to the cell body results in growth of the new axon from a dendrite, and cutting further out may not, we asked whether the initial response in the cell body was similar after both types of injury. A transcriptional reporter for axon injury signaling, puc-GFP, increased with similar timing and levels after proximal and distal axotomy. However, changes in dendritic microtubule polarity differed in response to the two types of injury, and were influenced by the presence of a scar at the distal axotomy site. CONCLUSIONS: We conclude that both ddaE and ddaC can regenerate axons either from the stump or a dendrite, and that there is some feedback between the two sites that modulates dendritic microtubule polarity.

Efficacy of Tat-conjugated ritonavir-loaded nanoparticles in reducing HIV-1 replication in monocyte-derived macrophages and cytocompatibility with macrophages and human neurons.

Human immunodeficiency virus (HIV)-1 targets mononuclear phagocytes (MP), which disseminate infection to organs such as brain, spleen and lymph. Thus MP, which include microglia, tissue macrophages and infiltrating monocyte-derived macrophages (MDM), are important target of anti-HIV-1 drug therapy. Most of the currently used antiretroviral drugs are effective in reducing viral loadin the periphery but cannot effectively eradicate infection from tissue reservoirs such as brain MP. HIV-1 infection of the central nervous system can lead to a wide variety of HIV-1-associated neurocognitive disorders. In this study, we demonstrate that ritonavir-loaded nanoparticles (RNPs) are effective in inhibiting HIV-1 infection of MDM. Reduced infection is observed in multiple read-out systems including reduction of cytopathic effects, HIV-1 p24 protein secretion and production of progeny virions. Furthermore, the RNPs retained antiretroviral efficacy after being removed from MDM cultures. As HIV-1-infected cells in the brain are likely to survive for a long period of time, both acute and chronic infection paradigms were evaluated. Tat-peptide-conjugated RNPs (Tat-RNP) were effective in both short-term and long-term HIV-1-infected MDM. Importantly, we confirm that delivery of RNPs, both with and without Tat-peptide conjugation, is toxic neither to MDM nor to neural cells, which may be bystander targets of the nanoformulations. In conclusion, Tat-NPs could be a safe and effective way of delivering anti-HIV-1 drugs for controlling viral replication occurring within brain MP.

Influence of scaffold size on bactericidal activity of nitric oxide-releasing silica nanoparticles.

A reverse microemulsion synthesis was used to prepare amine-functionalized silica nanoparticles of three distinct sizes (i.e., 50, 100, and 200 nm) with similar amine content. The resulting hybrid nanoparticles, consisting of N-(6-aminohexyl)aminopropyltrimethoxysilane and tetraethoxysilane, were highly monodisperse in size. N-Diazeniumdiolate nitric oxide (NO) donors were subsequently formed on secondary amines while controlling reaction conditions to keep the total amount of NO released constant for each particle size. The bactericidal efficacy of the NO-releasing nanoparticles against Pseudomonas aeruginosa increased with decreasing particle size. Additionally, smaller diameter nanoparticles were found to associate with the bacteria at a faster rate and to a greater extent than larger particles. Neither control (non-NO-releasing) nor NO-releasing particles exhibited toxicity toward L929 mouse fibroblasts at concentrations above their respective minimum bactericidal concentrations. This study represents the first investigation of the bactericidal efficacy of NO-releasing silica nanoparticles as a function of particle size.

Targeting anti-HIV drugs to the CNS.

The development of antiretroviral drugs over the past couple of decades has been commendable owing to the identification of several new targets within the overall HIV replication cycle. However, complete control over HIV/AIDS is yet to be achieved. This is because the current anti-HIV drugs, although effective in reducing plasma viral levels, cannot eradicate the virus completely from the body. This occurs because most anti-HIV drugs do not accumulate in certain cellular and anatomical reservoirs including the CNS. Insufficient delivery of anti-HIV drugs to the CNS is attributed to their low permeability across the BBB. Hence, low and sustained viral replication within the CNS continues even during prolonged antiretroviral drug therapy. Therefore, developing novel approaches that are targeted at enhancing the CNS delivery of anti-HIV drugs are required. In this review, we discuss the potential of nanocarriers and the role of cell-penetrating peptides in enhancing drug delivery to the CNS. Such drug delivery approaches could also lead to higher drug delivery to other cellular and anatomical reservoirs where the virus harbors than with conventional treatment, thus providing an effective therapy to eliminate the virus completely from the body.

Relevance of biophysical interactions of nanoparticles with a model membrane in predicting cellular uptake: study with TAT peptide-conjugated nanoparticles.

The aim of the study was to test the hypothesis that the biophysical interactions of the trans-activating transcriptor (TAT) peptide-conjugated nanoparticles (NPs) with a model cell membrane could predict the cellular uptake of the encapsulated therapeutic agent. To test the above hypothesis, the biophysical interactions of ritonavir-loaded poly(l-lactide) nanoparticles (RNPs), conjugated to either a TAT peptide (TAT-RNPs) or a scrambled TAT peptide (sc-TAT-RNPs), were studied with an endothelial cell model membrane (EMM) using a Langmuir film balance, and the corresponding human vascular endothelial cells (HUVECs) were used to study the uptake of the encapsulated therapeutic. Biophysical interactions were determined from the changes in surface pressure (SP) of the EMM as a function of time following interaction with NPs, and the compression isotherm (pi-A) of the EMM lipid mixture in the presence of NPs. In addition, the EMMs were transferred onto a silicon substrate following interactions with NPs using the Langmuir-Schaeffer (LS) technique. The transferred LS films were imaged by atomic force microscopy (AFM) to determine the changes in lipid morphology and to characterize the NP-membrane interactions. TAT-RNPs showed an increase in SP of the EMM, which was dependent upon the amount of the peptide bound to NPs and the concentration of NPs, whereas sc-TAT-RNPs and RNPs did not show any significant change in SP. The isotherm experiment showed a shift toward higher mean molecular area (mmA) in the presence of TAT-RNPs, indicating their interactions with the lipids of the EMM, whereas sc-TAT-RNPs and RNPs did not show any significant change. The AFM images showed condensation of the lipids following interaction with TAT-RNPs, indicating their penetration into the EMM, whereas RNPs did not cause any change. Surface analysis and 3-D AFM images of the EMM further confirmed penetration of TAT-RNPs into the EMM, whereas RNPs were seen anchored loosely to the membrane, and were significantly less in number than TAT-RNPs. We speculate that hydrophobic tyrosine of the TAT that forms the NP-interface drives the initial interactions of TAT-RNPs with the EMM, followed by electrostatic interactions with the anionic phospholipids of the membrane. In the case of sc-TAT-RNPs, hydrophilic arginine forms the NP-interface that does not interact with the EMM, despite having the similar cationic charge on these NPs as TAT-RNPs. TAT peptide alone did not show any change in SP, suggesting that the interaction occurs when the peptide is conjugated to a carrier system. HUVECs showed higher uptake of the drug with TAT-RNPs as compared to that with sc-TAT-RNPs or RNPs, suggesting that the biophysical interactions of NPs with cell membrane lipids play a role in cellular internalization of NPs. In conclusion, TAT peptide sequence and the amount of TAT conjugated to NPs significantly affect the biophysical interactions of NPs with the EMM, and these interactions correlate with the cellular delivery of the encapsulated drug. Biophysical interactions with a model membrane thus could be effectively used in developing efficient functionalized nanocarrier systems for drug delivery applications.