Tunable dual growth factor delivery from polyelectrolyte multilayer films

A promising strategy to accelerate joint implant integration and reduce recovery time and failure rates is to deliver a combination of certain growth factors to the integration site. There is a need to control the quantity of growth factors delivered at different times during the healing process to maximize efficacy. Polyelectrolyte multilayer (PEM) films, built using the layer-by-layer (LbL) technique, are attractive for releasing controlled amounts of potent growth factors over a sustained period. Here, we present PEM films that sequester physiological amounts of osteogenic rhBMP-2 (recombinant human bone morphogenetic protein - 2) and angiogenic rhVEGF[subscript 165] (recombinant human vascular endothelial growth factor) in different ratios in a degradable [poly(β-amino ester)/polyanion/growth factor/polyanion] LbL tetralayer repeat architecture where the biologic load scaled linearly with the number of tetralayers. No burst release of either growth factor was observed as the films degraded. The release of rhBMP-2 was sustained over a period of 2 weeks, while rhVEGF[subscript 165] eluted from the film over the first 8 days. Both growth factors retained their efficacy, as quantified with relevant in vitro assays. rhBMP-2 initiated a dose dependent differentiation cascade in MC3T3-E1S4 pre-osteoblasts while rhVEGF[subscript 165] upregulated HUVEC proliferation, and accelerated closure of a scratch in HUVEC cell cultures in a dose dependent manner. In vivo, the mineral density of ectopic bone formed de novo by rhBMP-2/rhVEGF[subscript 165] PEM films was approximately 33% higher than when only rhBMP-2 was introduced, with a higher trabecular thickness, which would indicate a decrease in the risk of osteoporotic fracture. Bone formed throughout the scaffold when both growth factors were released, which suggests more complete remodeling due to an increased local vascular network. This study demonstrates a promising approach to delivering precise doses of multiple growth factors for a variety of implant applications where control over spatial and temporal release profile of the biologic is desired.National Institutes of Health (U.S.) (National Institute on Aging Grant 5R01AG029601-04

Tissue integration of growth factor-eluting layer-by-layer polyelectrolyte multilayer coated implants

Drug eluting coatings that can direct the host tissue response to implanted medical devices have the potential to ameliorate both the medical and financial burden of complications from implantation. However, because many drugs useful in this arena are biologic in nature, a paucity of delivery strategies for biologics, including growth factors, currently limits the control that can be exerted on the implantation environment. Layer-by-Layer (LbL) polyelectrolyte multilayer films are highly attractive as ultrathin biologic reservoirs, due to the capability to conformally coat difficult geometries, the use of aqueous processing likely to preserve fragile protein function, and the tunability of incorporation and release profiles. Herein, we describe the first LbL films capable of microgram-scale release of the biologic Bone Morphogenetic Protein 2 (BMP-2), which is capable of directing the host tissue response to create bone from native progenitor cells. Ten micrograms of BMP-2 are released over a period of two weeks in vitro; less than 1% is released in the first 3 h (compared with commercial collagen matrices which can release up to 60% of BMP-2, too quickly to induce differentiation). BMP-2 released from LbL films retains its ability to induce bone differentiation in MC3T3 E1S4 pre-osteoblasts, as measured by induction of alkaline phosphatase and stains for calcium (via Alizarin Red) and calcium matrix (via Von Kossa). In vivo, BMP-2 film coated scaffolds were compared with film coated scaffolds lacking BMP-2. BMP-2 coatings implanted intramuscularly were able to initiate host progenitor cells to differentiate into bone, which matured and expanded from four to 9 weeks as measured by MicroCT and histology. Such LbL films represent new steps towards controlling and tuning host response to implanted medical devices, which may ultimately increase the success of implanted devices, provide alternative new approaches toward bone wound healing, and lay the foundation for development of a multi-therapeutic release coating.National Institutes of Health (U.S.) (Grant 1-R01-AG029601-01)Deshpande Center for Technological Innovation (Grant 009216-1)National Science Foundation (U.S.). Graduate Research Fellowshi

Pressure Overload in Mice With Haploinsufficiency of Striated Preferentially Expressed Gene Leads to Decompensated Heart Failure

Striated preferentially expressed gene (Speg) is a member of the myosin light chain kinase family of proteins. Constitutive Speg deficient (Speg(-/-)) mice develop a dilated cardiomyopathy, and the majority of these mice die in utero or shortly after birth. In the present study we assessed the importance of Speg in adult mice. Speg(-/-) mice that survived to adulthood, or adult striated muscle-specific Speg knockout mice (Speg-KO), demonstrated cardiac dysfunction and evidence of increased left ventricular (LV) internal diameter and heart to body weight ratio. To determine whether heterozygosity of Speg interferes with the response of the heart to pathophysiologic stress, Speg(+/-) mice were exposed to pressure overload induced by transverse aortic constriction (TAC). At baseline, Speg(+/+) and Speg(+/-) hearts showed no difference in cardiac function. However, 4 weeks after TAC, Speg(+/-) mice had a marked reduction in LV function. This defect was associated with an increase in LV internal diameter and enhanced heart weight to body weight ratio, compared with Speg(+/+) mice after TAC. The response of Speg(+/-) mice to pressure overload also included increased fibrotic deposition in the myocardium, disruption of transverse tubules, and attenuation in cell contractility, compared with Speg(+/+) mice. Taken together, these data demonstrate that Speg is necessary for normal cardiac function and is involved in the complex adaptation of the heart in response to TAC. Haploinsufficiency of Speg results in decompensated heart failure when exposed to pressure overload

Fine Mapping of the 1p36 Deletion Syndrome Identifies Mutation of PRDM16 as a Cause of Cardiomyopathy

Deletion 1p36 syndrome is recognized as the most common terminal deletion syndrome. Here, we describe the loss of a gene within the deletion that is responsible for the cardiomyopathy associated with monosomy 1p36, and we confirm its role in nonsyndromic left ventricular noncompaction cardiomyopathy (LVNC) and dilated cardiomyopathy (DCM). With our own data and publically available data from array comparative genomic hybridization (aCGH), we identified a minimal deletion for the cardiomyopathy associated with 1p36del syndrome that included only the terminal 14 exons of the transcription factor PRDM16 (PR domain containing 16), a gene that had previously been shown to direct brown fat determination and differentiation. Resequencing of PRDM16 in a cohort of 75 nonsyndromic individuals with LVNC detected three mutations, including one truncation mutant, one frameshift null mutation, and a single missense mutant. In addition, in a series of cardiac biopsies from 131 individuals with DCM, we found 5 individuals with 4 previously unreported nonsynonymous variants in the coding region of PRDM16. None of the PRDM16 mutations identified were observed in more than 6,400 controls. PRDM16 has not previously been associated with cardiac disease but is localized in the nuclei of cardiomyocytes throughout murine and human development and in the adult heart. Modeling of PRDM16 haploinsufficiency and a human truncation mutant in zebrafish resulted in both contractile dysfunction and partial uncoupling of cardiomyocytes and also revealed evidence of impaired cardiomyocyte proliferative capacity. In conclusion, mutation of PRDM16 causes the cardiomyopathy in 1p36 deletion syndrome as well as a proportion of nonsyndromic LVNC and DCM

HIF2alpha cooperates with RAS to promote lung tumorigenesis in mice.

Members of the hypoxia-inducible factor (HIF) family of transcription factors regulate the cellular response to hypoxia. In non-small cell lung cancer (NSCLC), high HIF2alpha levels correlate with decreased overall survival, and inhibition of either the protein encoded by the canonical HIF target gene VEGF or VEGFR2 improves clinical outcomes. However, whether HIF2alpha is causal in imparting this poor prognosis is unknown. Here, we generated mice that conditionally express both a nondegradable variant of HIF2alpha and a mutant form of Kras (KrasG12D) that induces lung tumors. Mice expressing both Hif2a and KrasG12D in the lungs developed larger tumors and had an increased tumor burden and decreased survival compared with mice expressing only KrasG12D. Additionally, tumors expressing both KrasG12D and Hif2a were more invasive, demonstrated features of epithelial- mesenchymal transition (EMT), and exhibited increased angiogenesis associated with mobilization of circulating endothelial progenitor cells. These results implicate HIF2alpha causally in the pathogenesis of lung cancer in mice, demonstrate in vivo that HIF2alpha can promote expression of markers of EMT, and define HIF2alpha as a promoter of tumor growth and progression in a solid tumor other than renal cell carcinoma. They further suggest a possible causal relationship between HIF2alpha and prognosis in patients with NSCLC

Consensus guidelines for the use and interpretation of angiogenesis assays

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference

Researching COVID to enhance recovery (RECOVER) tissue pathology study protocol: Rationale, objectives, and design.

ImportanceSARS-CoV-2 infection can result in ongoing, relapsing, or new symptoms or organ dysfunction after the acute phase of infection, termed Post-Acute Sequelae of SARS-CoV-2 (PASC), or long COVID. The characteristics, prevalence, trajectory and mechanisms of PASC are poorly understood. The objectives of the Researching COVID to Enhance Recovery (RECOVER) tissue pathology study (RECOVER-Pathology) are to: (1) characterize prevalence and types of organ injury/disease and pathology occurring with PASC; (2) characterize the association of pathologic findings with clinical and other characteristics; (3) define the pathophysiology and mechanisms of PASC, and possible mediation via viral persistence; and (4) establish a post-mortem tissue biobank and post-mortem brain imaging biorepository.MethodsRECOVER-Pathology is a cross-sectional study of decedents dying at least 15 days following initial SARS-CoV-2 infection. Eligible decedents must meet WHO criteria for suspected, probable, or confirmed infection and must be aged 18 years or more at the time of death. Enrollment occurs at 7 sites in four U.S. states and Washington, DC. Comprehensive autopsies are conducted according to a standardized protocol within 24 hours of death; tissue samples are sent to the PASC Biorepository for later analyses. Data on clinical history are collected from the medical records and/or next of kin. The primary study outcomes include an array of pathologic features organized by organ system. Causal inference methods will be employed to investigate associations between risk factors and pathologic outcomes.DiscussionRECOVER-Pathology is the largest autopsy study addressing PASC among US adults. Results of this study are intended to elucidate mechanisms of organ injury and disease and enhance our understanding of the pathophysiology of PASC