Development of a nanoparticle-based tendon-targeting drug delivery system to pharmacologically modulate tendon healing.

Satisfactory healing following acute tendon injury is marred by fibrosis. Despite the high frequency of tendon injuries and poor outcomes, there are no pharmacological therapies in use to enhance the healing process. Moreover, systemic treatments demonstrate poor tendon homing, limiting the beneficial effects of potential tendon therapeutics. To address this unmet need, we leveraged our existing tendon healing spatial transcriptomics dataset and identified an area enriched for expression of Acp5 (TRAP) and subsequently demonstrated robust TRAP activity in the healing tendon. This unexpected finding allowed us to refine and apply our existing TRAP binding peptide (TBP) functionalized nanoparticle (NP) drug delivery system (DDS) to facilitate improved delivery of systemic treatments to the healing tendon. To demonstrate the translational potential of this DDS, we delivered niclosamide (NEN), an S100a4 inhibitor. While systemic delivery of free NEN did not alter healing, TBP-NPNEN enhanced both functional and mechanical recovery, demonstrating the translational potential of this approach to enhance the tendon healing process.

Development of a Nanoparticle-Based Tendon-Targeting Drug Delivery System to Pharmacologically Modulate Tendon Healing.

Tendon regeneration following acute injury is marred by a fibrotic healing response that prevents complete functional recovery. Despite the high frequency of tendon injuries and the poor outcomes, including functional deficits and elevated risk of re-injury, there are currently no pharmacological therapies in clinical use to enhance the healing process. Several promising pharmacotherapies have been identified; however, systemic treatments lack tendon specificity, resulting in poor tendon biodistribution and perhaps explaining the largely limited beneficial effects of these treatments on the tendon healing process. To address this major unmet need, we leveraged our existing spatial transcriptomics dataset of the tendon healing process to identify an area of the healing tendon that is enriched for expression of Acp5. Acp5 encodes tartrate-resistant acid phosphatase (TRAP), and we demonstrate robust TRAP activity in the healing tendon. This unexpected finding allowed us to refine and apply our existing TRAP binding peptide (TBP) functionalized nanoparticle (NP) drug delivery system (DDS) to facilitate improved delivery of systemic treatments to the healing tendon. To demonstrate the translational potential of this drug delivery system, we delivered the S100a4 inhibitor, Niclosamide to the healing tendon. We have previously shown that genetic knockdown of S100a4 enhances tendon healing. While systemic delivery of Niclosamide did not affect the healing process, relative to controls, TBP-NP delivery of Niclosamide enhanced both functional and mechanical outcome measures. Collectively, these data identify a novel tendon-targeting drug delivery system and demonstrate the translational potential of this approach to enhance the tendon healing process.

HSF-1: Guardian of the Proteome Through Integration of Longevity Signals to the Proteostatic Network.

Discoveries made in the nematode Caenorhabditis elegans revealed that aging is under genetic control. Since these transformative initial studies, C. elegans has become a premier model system for aging research. Critically, the genes, pathways, and processes that have fundamental roles in organismal aging are deeply conserved throughout evolution. This conservation has led to a wealth of knowledge regarding both the processes that influence aging and the identification of molecular and cellular hallmarks that play a causative role in the physiological decline of organisms. One key feature of age-associated decline is the failure of mechanisms that maintain proper function of the proteome (proteostasis). Here we highlight components of the proteostatic network that act to maintain the proteome and how this network integrates into major longevity signaling pathways. We focus in depth on the heat shock transcription factor 1 (HSF1), the central regulator of gene expression for proteins that maintain the cytosolic and nuclear proteomes, and a key effector of longevity signals.

Bone Marrow Microenvironment-On-Chip for Culture of Functional Hematopoietic Stem Cells.

Hematopoiesis takes place in the bone marrow and is supported by a complex cellular and molecular network in the bone marrow microenvironment. Commonly used models of the human bone marrow microenvironment include murine models and two-dimensional and three-dimensional tissue cultures. While these model systems have led to critical advances in the field, they fail to recapitulate many aspects of the human bone marrow. This has limited our understanding of human bone marrow pathophysiology and has led to deficiencies in therapy for many bone marrow pathologies such as bone marrow failure syndromes and leukemias. Therefore, we have developed a modular murine bone marrow microenvironment-on-chip using a commercially available microfluidic platform. This model includes a vascular channel separated from the bone marrow channel by a semi-porous membrane and incorporates critical components of the bone marrow microenvironment, including osteoblasts, endothelial cells, mesenchymal stem cells, and hematopoietic stem and progenitor cells. This system is capable of maintaining functional hematopoietic stem cells in vitro for at least 14 days at frequencies similar to what is found in the primary bone marrow. The modular nature of this system and its accessibility will allow for acceleration of our understanding of the bone marrow.

From the niche to malignant hematopoiesis and back: reciprocal interactions between leukemia and the bone marrow microenvironment.

The bone marrow microenvironment (BMME) regulates hematopoiesis through a complex network of cellular and molecular components. Hematologic malignancies reside within, and extensively interact with, the same BMME. These interactions consequently alter both malignant and benign hematopoiesis in multiple ways, and can encompass initiation of malignancy, support of malignant progression, resistance to chemotherapy, and loss of normal hematopoiesis. Herein, we will review supporting studies for interactions of the BMME with hematologic malignancies and discuss challenges still facing this exciting field of research. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Reduction of leukemic burden via bone-targeted nanoparticle delivery of an inhibitor of C-chemokine (C-C motif) ligand 3 (CCL3) signaling.

Leukemias are challenging diseases to treat due, in part, to interactions between leukemia cells and the bone marrow microenvironment (BMME) that contribute significantly to disease progression. Studies have shown that leukemic cells secrete C-chemokine (C-C motif) ligand 3 (CCL3), to disrupt the BMME resulting in loss of hematopoiesis and support of leukemic cell survival and proliferation. In this study, a murine model of blast crisis chronic myelogenous leukemia (bcCML) that expresses the translocation products BCR/ABL and Nup98/HoxA9 was used to determine the role of CCL3 in BMME regulation. Leukemic cells derived from CCL3-/- mice were shown to minimally engraft in a normal BMME, thereby demonstrating that CCL3 signaling was necessary to recapitulate bcCML disease. Further analysis showed disruption in hematopoiesis within the BMME in the bcCML model. To rescue the altered BMME, therapeutic inhibition of CCL3 signaling was investigated using bone-targeted nanoparticles (NP) to deliver Maraviroc, an inhibitor of C-C chemokine receptor type 5 (CCR5), a CCL3 receptor. NP-mediated Maraviroc delivery partially restored the BMME, significantly reduced leukemic burden, and improved survival. Overall, our results demonstrate that inhibiting CCL3 via CCR5 antagonism is a potential therapeutic approach to restore normal hematopoiesis as well as reduce leukemic burden within the BMME.

3D iPSC modeling of the retinal pigment epithelium-choriocapillaris complex identifies factors involved in the pathology of macular degeneration.

The retinal pigment epithelium (RPE)-choriocapillaris (CC) complex in the eye is compromised in age-related macular degeneration (AMD) and related macular dystrophies (MDs), yet in vitro models of RPE-CC complex that enable investigation of AMD/MD pathophysiology are lacking. By incorporating iPSC-derived cells into a hydrogel-based extracellular matrix, we developed a 3D RPE-CC model that recapitulates key features of both healthy and AMD/MD eyes and provides modular control over RPE and CC layers. Using this 3D RPE-CC model, we demonstrated that both RPE- and mesenchyme-secreted factors are necessary for the formation of fenestrated CC-like vasculature. Our data show that choroidal neovascularization (CNV) and CC atrophy occur in the absence of endothelial cell dysfunction and are not necessarily secondary to drusen deposits underneath RPE cells, and CC atrophy and/or CNV can be initiated systemically by patient serum or locally by mutant RPE-secreted factors. Finally, we identify FGF2 and matrix metalloproteinases as potential therapeutic targets for AMD/MDs.

A human model of Batten disease shows role of CLN3 in phagocytosis at the photoreceptor-RPE interface.

Mutations in CLN3 lead to photoreceptor cell loss in CLN3 disease, a lysosomal storage disorder characterized by childhood-onset vision loss, neurological impairment, and premature death. However, how CLN3 mutations cause photoreceptor cell death is not known. Here, we show that CLN3 is required for phagocytosis of photoreceptor outer segment (POS) by retinal pigment epithelium (RPE) cells, a cellular process essential for photoreceptor survival. Specifically, a proportion of CLN3 in human, mouse, and iPSC-RPE cells localized to RPE microvilli, the site of POS phagocytosis. Furthermore, patient-derived CLN3 disease iPSC-RPE cells showed decreased RPE microvilli density and reduced POS binding and ingestion. Notably, POS phagocytosis defect in CLN3 disease iPSC-RPE cells could be rescued by wild-type CLN3 gene supplementation. Altogether, these results illustrate a novel role of CLN3 in regulating POS phagocytosis and suggest a contribution of primary RPE dysfunction for photoreceptor cell loss in CLN3 disease that can be targeted by gene therapy.

Environmental stress impairs photoreceptor outer segment (POS) phagocytosis and degradation and induces autofluorescent material accumulation in hiPSC-RPE cells.

Retinal pigment epithelium (RPE) cell dysfunction is central to the pathogenesis of age-related macular degeneration (AMD), a leading cause of adult blindness. Aging, the single biggest risk factor for AMD development, favors increase in RPE autofluorescent material due to accumulation of POS-digestion by-products through lysosomal dysfunction and impaired POS degradation. Apart from aging, environmental agents affect lysosomal function in multiple model systems and are implicated in AMD. Iron (Fe) overload and cigarette smoke exposure are the two environmental factors that are known to affect the lysosomal pathway and impact RPE cell health. However, the impact of Fe and cigarette smoke, on POS processing and its consequence for autofluorescent material accumulation in human RPE cells are yet to be established. Human induced pluripotent stem cell (hiPSC)-derived RPE, which phagocytoses and degrades POS in culture and can be derived from control individuals (no history/susceptibility for retinal disease), provides a model system to investigate the singular effect of excess Fe and/or cigarette smoke on POS processing by RPE cells. Using at least three distinct control hiPSC lines, we show that, compared to untreated hiPSC-RPE cells, POS uptake is reduced in both Fe (ferric ammonium citrate or FAC) and FAC + CSE (cigarette smoke extract)-treated hiPSC-RPE cells. Furthermore, exposure of hiPSC-RPE cultures to FAC + CSE leads to reduced levels of active cathepsin-D (CTSD), a lysosomal enzyme involved in POS processing, and causes delayed degradation of POS. Notably, delayed degradation of POS over time (2 weeks) in hiPSC-RPE cells exposed to Fe and CSE was sufficient to increase autofluorescent material build-up in these cells. Given that inefficient POS processing-mediated autofluorescent material accumulation in RPE cells has already been linked to AMD development, our results implicate a causative role of environmental agents, like Fe and cigarette smoke, in AMD.

Gemcitabine plus vinorelbine versus vinorelbine monotherapy in patients with metastatic breast cancer previously treated with anthracyclines and taxanes: final results of the phase III Spanish Breast Cancer Research Group (GEICAM) trial.

BACKGROUND: We aimed to compare the additional benefit of gemcitabine when combined with vinorelbine above that of standard vinorelbine treatment in patients with metastatic breast cancer. METHODS: In this phase III, multicentre, open-label, randomised study, 252 women with locally recurrent and metastatic breast cancer who had been pretreated with anthracyclines and taxanes were randomly assigned single-agent vinorelbine (30 mg/m(2), days 1 and 8) or gemcitabine plus vinorelbine (1200/30 mg/m(2), days 1 and 8). Both study treatments were administered intravenously every 21 days until disease progression, unacceptable toxic effects, or stoppage at the request of investigator or patient. The primary endpoint was median progression-free survival. Secondary objectives included assessments of response rate, disease duration, overall survival, and characterisation of the toxicity profiles of both regimens. This study is registered with ClinicalTrials.gov, number NCT00128310. FINDINGS: Between 2001 and 2005, 252 women were recruited and randomised for treatment. One of these patients was ineligible. Prognostic factors were well balanced between treatment groups (median number of metastatic sites in combination group 2 (range 0-5) and in vinorelbine group 2 (range 1-6); visceral disease in 76% and 75% of patients, respectively). Median progression-free survival was 6.0 months (95% CI 4.8-7.1) for patients given gemcitabine plus vinorelbine and 4.0 months (2.9-5.1) for those assigned vinorelbine; there was 1.9 months of difference (hazard ratio 0.66 [0.50-0.88]; p=0.0028). Overall survival was 15.9 months (12.6-19.1) for the gemcitabine plus vinorelbine group and 16.4 months (11.6-21.0) for the vinorelbine group; there was 0.5 months of difference (hazard ratio 1.04 [0.78-1.39]; p=0.8046). Objective response rates were 36% for patients assigned gemcitabine plus vinorelbine (n=45) and 26% for those assigned vinorelbine (n=33) (p=0.093). Grade 3 or 4 neutropenia was reported in 75 (61% [52-70]) of the participants assigned gemcitabine plus vinorelbine, compared with 55 (44% [35-53]) of those assigned vinorelbine alone (p=0.0074). Febrile neutropenia occurred in 13 (11%) of those assigned gemcitabine plus vinorelbine, and in seven (6%) of those assigned vinorelbine alone (p=0.15). Incidences of grade 3 or 4 non-haematological toxic effects were similar between the two treatment groups. INTERPRETATION: Patients with metastatic breast cancer assigned gemcitabine and vinorelbine had better progression-free survival compared with those assigned vinorelbine alone. However, this finding did not translate into a difference in overall survival. Although toxicity was manageable, patients in the combined group had more haematological toxic effects. These factors should be taken into account when deciding which chemotherapy patients should receive.