Nina Mann

Precise engineering of targeted nanoparticles by using self-assembled biointegrated block copolymers

There has been progressively heightened interest in the development of targeted nanoparticles (NPs) for differential delivery and controlled release of drugs. Despite nearly three decades of research, approaches to reproducibly formulate targeted NPs with the optimal biophysicochemical properties have remained elusive. A central challenge has been defining the optimal interplay of parameters that confer molecular targeting, immune evasion, and drug release to overcome the physiological barriers in vivo. Here, we report a strategy for narrowly changing the biophysicochemical properties of NPs in a reproducible manner, thereby enabling systematic screening of optimally formulated drug-encapsulated targeted NPs. NPs were formulated by the self-assembly of an amphiphilic triblock copolymer composed of end-to-end linkage of poly(lactic-co-glycolic-acid) (PLGA), polyethyleneglycol (PEG), and the A10 aptamer (Apt), which binds to the prostate-specific membrane antigen (PSMA) on the surface of prostate cancer (PCa) cells, enabling, respectively, controlled drug release, “stealth” properties for immune evasion, and cell-specific targeting. Fine-tuning of NP size and drug release kinetics was further accomplished by controlling the copolymer composition. By using distinct ratios of PLGA-b-PEG-b-Apt triblock copolymer with PLGA-b-PEG diblock copolymer lacking the A10 Apt, we developed a series of targeted NPs with increasing Apt densities that inversely affected the amount of PEG exposure on NP surface and identified the narrow range of Apt density when the NPs were maximally targeted and maximally stealth, resulting in most efficient PCa cell uptake in vitro and in vivo. This approach may contribute to further development of targeted NPs as highly selective and effective therapeutic modalities.

Can Exercise Teach Us How to Treat Heart Disease

Exercise is one of the mainstay clinical interventions for the prevention and treatment of cardiovascular disease. Not only does exercise reduce cardiovascular risk factors, such as diabetes and hypertension, thereby helping prevent heart disease, it also appears to improve the functional status and outcomes in patients with existing heart disease.1-6 The cardiovascular benefits of exercise are multifactorial, and include important systemic effects (Figure 1) on skeletal muscle, the peripheral vasculature, and metabolism, as well as beneficial alterations within the myocardium itself.7, 8 Figure 1 Overview of the systemic and cardiac-specific effects of exercise Many current pharmacological treatments for cardiovascular disease are targeted towards inhibiting the adverse remodeling process associated with pathological stress. Specifically, they focus on abrogating the pathological hypertrophy, fibrosis, electrical remodeling, and cavity dilatation that accompany disease states such as longstanding hypertension and myocardial infarction.9-11 Interestingly, exercise, like many of these pathological stimuli, can also induce cardiac and cardiomyocyte hypertrophy. However, growing evidence suggests that such physiological remodeling, rather than leading to adverse sequelae, may actually be cardioprotective and that activating pathways associated with exercise can help to prevent and treat cardiovascular disease.8, 12, 13 In this review, we discuss recent advances in our understanding of the cellular and molecular mechanisms (Figure 2) that mediate the cardiac response to exercise, including cardiomyocyte hypertrophy and renewal, vascular remodeling, and alterations in calcium handling and metabolism. In addition to classical signaling mechanisms and transcriptional networks, we describe the role of secreted molecules and miRNAs. Finally, an emerging theme is that pathways that are either regulated by exercise or that mediate the hearts response to exercise often also have the potential to mitigate or even reverse cardiac disease. Thus, we suggest that understanding the effects of exercise more fully may provide useful biological insights and open the door to new therapeutic approaches aimed at restoring cardiovascular health. Figure 2 Key signaling pathways involved in mediating exercise-induced cardiac remodeling Physiological Cardiac Remodeling Exercise is perhaps one of the cheapest — and most effective — interventions for reducing the morbidity and mortality of cardiovascular disease.14 In fact, as little as 45-75 minutes of brisk walking each week appears to reduce the relative risk for adverse cardiac events.15, 16 Additionally, exercise-based cardiac rehabilitation is recommended by the American Heart Association (AHA) as one of the mainstay interventions following acute myocardial infarction (MI), with maximal benefit derived from early initiation of exercise (as early as one week post MI-hospital discharge) and from increased duration of exercise rehabilitation.1-3 Multiple studies have also demonstrated a dose-response relationship between exercise and cardiovascular benefit, but the shape of that curve, and the optimal dosage, intensity, frequency, and duration of exercise remain incompletely defined.15, 17, 18 The health benefits of exercise are multifactorial. Studies have demonstrated that physical activity is effective in reducing adipocyte mass and body mass index as well as positively affecting insulin sensitivity, glucose uptake by skeletal muscle, and cholesterol profiles.19 Physical activity — aerobic exercise, in particular — has also been associated with beneficial changes in both the systemic and coronary vasculature, including enhanced endothelial-mediated vasodilation, improved arterial compliance, and reductions in both systolic and diastolic blood pressure.20-22 Although these global effects of exercise are all implicated in improving cardiovascular health, here, we will focus primarily on the cardiac-specific effects of exercise. Cardiac Growth The heart has considerable plasticity9 and its capacity to hypertrophy in response to pathological stimuli, such as hypertension, aortic stenosis, or genetic mutations, is familiar to clinical cardiologists. However, a robust hypertrophic response is also seen with physiological stimuli, including exercise, pregnancy, and postnatal growth. Endurance exercise and pregnancy, for example, can induce up to a 20% increase in left ventricular (LV) mass, while, even more impressively, the hearts of Burmese pythons can grow by up to 40% following meals.23, 24 The cellular response to growth signals is often categorized as either hypertrophic — an increase in cell size — or hyperplastic — an increase in cell number. The adult heart has traditionally been viewed as capable only of hypertrophic growth; however, recent data from animal models and human studies suggest that the heart also has a limited capacity to generate new cardiomyocytes from progenitor cells and existing cardiomyocytes.25-27 In clinical practice, it is impossible, with current imaging modalities, to distinguish between these two distinct mechanisms of growth when characterizing cardiac hypertrophy. However, animal studies suggest that an increase in both cardiomyocyte size and number may contribute to heart growth in response to pathological and physiological stimuli.12, 28 Exercise-induced cardiac remodeling is the prototypical example of physiological cardiac growth, and the hypertrophic response to exercise can broadly be described as either concentric or eccentric hypertrophy, or a combination of the two. Isometric exercises — strength training activities like weight lifting — lead to transient increases in systemic vascular resistance, thereby increasing afterload and predominantly produce concentric hypertrophy, in which sarcomere fibers are added in parallel with subsequent thickening of the ventricular wall. Endurance — or isotonic — exercise, such as swimming and running, present a volume challenge to the heart and tend to result in eccentric hypertrophy, with increased preload and end-diastolic volume.29, 30 Cardiac MRI studies have suggested that isometric exercises induce minimal changes in right ventricular (RV) structure and function, while isotonic exercises lead to a balanced biventricular hypertrophy with symmetric enlargement of both the right and left ventricles.31 Cardiomyocyte hypertrophy is likely the dominant contributor to exercise-induced heart growth, and studies have reported an increase in cardiomyocyte size by up to 17-32% following exercise training.32 As noted above, however, recent work suggests that exercise also induces markers of cardiomyocyte proliferation, although the fate and contribution of these newly formed cells remains to be established.12 A recently described model for studying physiological remodeling is the Burmese python, which demonstrates an impressive increase in cardiac size — up to 40% — following meals, which regresses over the subsequent 28 days.24 Emerging data suggest that this increase in heart size is primarily a hypertrophic, rather than hyperplastic, process, that it is not associated with the characteristic changes seen in pathological cardiac growth such as fibrosis and upregulation of the fetal gene program.33 This lends support to the idea that physiological hypertrophy is primarily an adaptive and beneficial process. Interestingly, new evidence suggests that some of these postprandial cardiac growth effects are mediated by secreted lipids,33 which will be discussed in more detail below. It should be acknowledged that the clinical relevance of post-prandial changes in the Burmese python remain unclear. Interestingly, the combination of fatty acids identified in python serum also induced cardiomyocyte hypertrophy in mice.33

A Dominant Mutation in Nuclear Receptor Interacting Protein 1 Causes Urinary Tract Malformations via Dysregulation of Retinoic Acid Signaling

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of CKD in the first three decades of life. However, for most patients with CAKUT, the causative mutation remains unknown. We identified a kindred with an autosomal dominant form of CAKUT. By whole-exome sequencing, we identified a heterozygous truncating mutation (c.279delG, p.Trp93fs*) of the nuclear receptor interacting protein 1 gene (NRIP1) in all seven affected members. NRIP1 encodes a nuclear receptor transcriptional cofactor that directly interacts with the retinoic acid receptors (RARs) to modulate retinoic acid transcriptional activity. Unlike wild-type NRIP1, the altered NRIP1 protein did not translocate to the nucleus, did not interact with RARα, and failed to inhibit retinoic acid-dependent transcriptional activity upon expression in HEK293 cells. Notably, we also showed that treatment with retinoic acid enhanced NRIP1 binding to RARα RNA in situ hybridization confirmed Nrip1 expression in the developing urogenital system of the mouse. In explant cultures of embryonic kidney rudiments, retinoic acid stimulated Nrip1 expression, whereas a pan-RAR antagonist strongly reduced it. Furthermore, mice heterozygous for a null allele of Nrip1 showed a CAKUT-spectrum phenotype. Finally, expression and knockdown experiments in Xenopus laevis confirmed an evolutionarily conserved role for NRIP1 in renal development. These data indicate that dominant NRIP1 mutations can cause CAKUT by interference with retinoic acid transcriptional signaling, shedding light on the well documented association between abnormal vitamin A levels and renal malformations in humans, and suggest a possible gene-environment pathomechanism in this disease.

Exome sequencing in Jewish and Arab patients with rhabdomyolysis reveals single-gene etiology in 43% of cases

BackgroundRhabdomyolysis is a clinical emergency that may cause acute kidney injury (AKI). It can be acquired or due to monogenic mutations. Around 60 different rare monogenic forms of rhabdomyolysis have been reported to date. In the clinical setting, identifying the underlying molecular diagnosis is challenging due to nonspecific presentation, the high number of causative genes, and current lack of data on the prevalence of monogenic forms.MethodsWe employed whole exome sequencing (WES) to reveal the percentage of rhabdomyolysis cases explained by single-gene (monogenic) mutations in one of 58 candidate genes. We investigated a cohort of 21 unrelated families with rhabdomyolysis, in whom no underlying etiology had been previously established.ResultsUsing WES, we identified causative mutations in candidate genes in nine of the 21 families (43%). We detected disease-causing mutations in eight of 58 candidate genes, grouped into the following categories: (1) disorders of fatty acid metabolism (CPT2), (2) disorders of glycogen metabolism (PFKM and PGAM2), (3) disorders of abnormal skeletal muscle relaxation and contraction (CACNA1S, MYH3, RYR1 and SCN4A), and (4) disorders of purine metabolism (AHCY).ConclusionsOur findings demonstrate a very high detection rate for monogenic etiologies using WES and reveal broad genetic heterogeneity for rhabdomyolysis. These results highlight the importance of molecular genetic diagnostics for establishing an etiologic diagnosis. Because these patients are at risk for recurrent episodes of rhabdomyolysis and subsequent risk for AKI, WES allows adequate prophylaxis and treatment for these patients and their family members and enables a personalized medicine approach.

A homozygous missense variant in VWA2, encoding an interactor of the Fraser-complex, in a patient with vesicoureteral reflux

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause (40–50%) of chronic kidney disease (CKD) in children. About 40 monogenic causes of CAKUT have so far been discovered. To date less than 20% of CAKUT cases can be explained by mutations in these 40 genes. To identify additional monogenic causes of CAKUT, we performed whole exome sequencing (WES) and homozygosity mapping (HM) in a patient with CAKUT from Indian origin and consanguineous descent. We identified a homozygous missense mutation (c.1336C>T, p.Arg446Cys) in the gene Von Willebrand factor A domain containing 2 (VWA2). With immunohistochemistry studies on kidneys of newborn (P1) mice, we show that Vwa2 and Fraser extracellular matrix complex subunit 1 (Fras1) co-localize in the nephrogenic zone of the renal cortex. We identified a pronounced expression of Vwa2 in the basement membrane of the ureteric bud (UB) and derivatives of the metanephric mesenchyme (MM). By applying in vitro assays, we demonstrate that the Arg446Cys mutation decreases translocation of monomeric VWA2 protein and increases translocation of aggregated VWA2 protein into the extracellular space. This is potentially due to the additional, unpaired cysteine residue in the mutated protein that is used for intermolecular disulfide bond formation. VWA2 is a known, direct interactor of FRAS1 of the Fraser-Complex (FC). FC-encoding genes and interacting proteins have previously been implicated in the pathogenesis of syndromic and/or isolated CAKUT phenotypes in humans. VWA2 therefore constitutes a very strong candidate in the search for novel CAKUT-causing genes. Our results from in vitro experiments indicate a dose-dependent neomorphic effect of the Arg446Cys homozygous mutation in VWA2.

Whole-Exome Sequencing Identifies Causative Mutations in Families with Congenital Anomalies of the Kidney and Urinary Tract

BACKGROUND Congenital anomalies of the kidney and urinary tract (CAKUT) are the most prevalent cause of kidney disease in the first three decades of life. Previous gene panel studies showed monogenic causation in up to 12% of patients with CAKUT. METHODS We applied whole-exome sequencing to analyze the genotypes of individuals from 232 families with CAKUT, evaluating for mutations in single genes known to cause human CAKUT and genes known to cause CAKUT in mice. In consanguineous or multiplex families, we additionally performed a search for novel monogenic causes of CAKUT. RESULTS In 29 families (13%), we detected a causative mutation in a known gene for isolated or syndromic CAKUT that sufficiently explained the patients CAKUT phenotype. In three families (1%), we detected a mutation in a gene reported to cause a phenocopy of CAKUT. In 15 of 155 families with isolated CAKUT, we detected deleterious mutations in syndromic CAKUT genes. Our additional search for novel monogenic causes of CAKUT in consanguineous and multiplex families revealed a potential single, novel monogenic CAKUT gene in 19 of 232 families (8%). CONCLUSIONS We identified monogenic mutations in a known human CAKUT gene or CAKUT phenocopy gene as the cause of disease in 14% of the CAKUT families in this study. Whole-exome sequencing provides an etiologic diagnosis in a high fraction of patients with CAKUT and will provide a new basis for the mechanistic understanding of CAKUT.

GEOMETRY OF TRICUSPID VALVE INFLOW CORRELATES WITH VENTILATORY EFFICIENCY AND PEAK OXYGEN PULSE IN ADULTS WITH SYSTEMIC RIGHT VENTRICLES AND TRANSPOSITION OF THE GREAT ARTERIES

Exercise test variables including ventilatory efficiency (VE/VCO2 slope) and peak oxygen uptake (pVO2) correlate with mortality in adults with congenital heart disease. O2 pulse (VO2 divided by heart rate) is used as a surrogate for stroke volume. However, correlation of these exercise values with