Angiotensin II receptor type 1 blockade regulates Klotho expression to induce TSC2-deficient cell death.

Lymphangioleiomyomatosis (LAM) is a multisystem disease occurring in women of child-bearing age manifested by uncontrolled proliferation of smooth muscle-like "LAM" cells in the lungs. LAM cells bear loss-of-function mutations in tuberous sclerosis complex (TSC) genes TSC1 and/or TSC2, causing hyperactivation of the proliferation promoting mammalian/mechanistic target of Rapamycin complex 1 pathway. Additionally, LAM-specific active renin-angiotensin system (RAS) has been identified in LAM nodules, suggesting this system potentially contributes to neoplastic properties of LAM cells; however, the role of this renin-angiotensin signaling is unclear. Here, we report that TSC2-deficient cells are sensitive to the blockade of angiotensin II receptor type 1 (Agtr1). We show that treatment of these cells with the AGTR1 inhibitor losartan or silencing of the Agtr1 gene leads to increased cell death in vitro and attenuates tumor progression in vivo. Notably, we found the effect of Agtr1 blockade is specific to TSC2-deficient cells. Mechanistically, we demonstrate that cell death induced by Agtr1 inhibition is mediated by an increased expression of Klotho. In TSC2-deficient cells, we showed overexpression of Klotho or treatment with recombinant (soluble) Klotho mirrored the cytocidal effect of angiotensin blockade. Furthermore, Klotho treatment decreased the phosphorylation of AKT, potentially leading to this cytocidal effect. Conversely, silencing of Klotho rescued TSC2-deficient cells from cell death induced by Agtr1 inhibition. Therefore, we conclude that Agtr1 and Klotho are important for TSC2-deficient cell survival. These findings further illuminate the role of the RAS in LAM and the potential of targeting Agtr1 inhibition in TSC2-deficient cells.

The role of the TSC Alliance in advancing therapy development: a patient organization perspective.

Tuberous sclerosis complex (TSC) is a genetic disease leading to malformations, or tubers, in the cerebral cortex and growth of tumors, most frequently in the brain, heart, kidneys, skin, and lungs. Changes in the brain caused by TSC usually have the biggest negative impact on quality of life. Approximately 85% of individuals with TSC have epilepsy, and TSC-associated neuropsychiatric disorders (TAND) affect nearly all individuals with TSC in some way. TSC Alliance's research strategy is built upon both funding and catalyzing research. Through grants, the organization provides funding directly to researchers through a competitive application process. The organization has also built a set of resources available to researchers worldwide, including a Natural History Database, Biosample Repository, and Preclinical Consortium. These resources catalyze research because they are available to qualified academic or industry researchers around the world, enabling an almost unlimited number of scientists to access data and resources to enable and accelerate research on TSC. This research strategy continues to be shaped by the needs and priorities of the TSC community, working toward a future where everyone affected by TSC can live their fullest lives.

The role of the TSC Alliance in advancing therapy development: a patient organization perspective Finding a new treatment for any disease is a long and expensive process, and it can be even more challenging for a rare disease such as tuberous sclerosis complex (TSC). To encourage research on TSC and speed up the process developing new treatments, the TSC Alliance established a research strategy based upon the priorities of people living with TSC. TSC community members best know how the disease negatively affects their lives. Equally importantly, the TSC community is a necessary partner for any researcher or company who wants to bring forward a potential new treatment. The TSC Alliance awards research grants to individual researchers who are at early stages of their careers. We also collaborate with many researchers and healthcare providers, and with the TSC community, to build shared resources. These resources include data from medical records and biological samples, such as blood and tissue samples, which are shared with researchers around the world for a wide range of projects related to TSC. We also collaborate with researchers from academic laboratories and the pharmaceutical or biotech industry to test potential new drugs or other therapies in animals, which is required before new therapies can be tested in humans. Before and during human testing in clinical trials, we help researchers design a trial that is both meaningful to the TSC community and not overly burdensome to participants. As new therapies become available, the TSC Alliance educates the TSC community and advocates for patient access to new therapies. Over time, as more is learned about how best to monitor and treat people with TSC, the organization convenes a conference of TSC experts to update clinical consensus guidelines to guide improved treatment of this rare disease.

Circulating biomarkers of kidney angiomyolipoma and cysts in tuberous sclerosis complex patients.

Patients with tuberous sclerosis complex (TSC) develop multi-organ disease manifestations, with kidney angiomyolipomas (AML) and cysts being one of the most common and deadly. Early and regular AML/cyst detection and monitoring are vital to lower TSC patient morbidity and mortality. However, the current standard of care involves imaging-based methods that are not designed for rapid screening, posing challenges for early detection. To identify potential diagnostic screening biomarkers of AML/cysts, we performed global untargeted metabolomics in blood samples from 283 kidney AML/cyst-positive or -negative TSC patients using mass spectrometry. We identified 7 highly sensitive chemical features, including octanoic acid, that predict kidney AML/cysts in TSC patients. Patients with elevated octanoic acid have lower levels of very long-chain fatty acids (VLCFAs), suggesting that dysregulated peroxisome activity leads to overproduction of octanoic acid via VLCFA oxidation. These data highlight AML/cysts blood biomarkers for TSC patients and offers valuable metabolic insights into the disease.

Data evaluating triamcinolone acetonide and triamcinolone hexacetonide loaded poly(δ-valerolactone-co-allyl-δ-valerolactone) microparticles.

Advanced drug delivery strategies can be used to enhance the therapeutic effectiveness of locally delivered corticosteroids. Poly(δ-valerolactone-co-allyl-δ-valerolactone) microparticles (PVL-co-PAVL MPs) were evaluated for delivery of two corticosteroids, triamcinolone acetonide and triamcinolone hexacetonide. PVL-co-PAVL MPs were prepared using a modified oil-in-water emulsification method, followed by a UV-initiated cross-linking process. The resulting PVL-co-PAVL MPs were purified with an excess amount of water and then acetone to remove residual surfactant, cross-linker, and catalyst before lyophilization. Triamcinolone acetonide and triamcinolone hexacetonide were independently loaded into the resulting PVL-co-PAVL MPs via a post-loading swelling-equilibrium method. The drug-loaded MPs were characterized in terms of drug loading (determined by high-performance liquid chromatography, HPLC), thermal properties (determined by differential scanning calorimetry, DSC), and in vitro drug release kinetics (with quantification of drug using HPLC) to better understand the suitability of PVL-co-PAVL MPs for delivery of corticosteroids. These data demonstrate the potential of PVL-co-PAVL MPs as a promising drug delivery platform for the sustained release of corticosteroids. Raw data have been made available on Mendeley Data. Additional details on PVL-co-PAVL MPs were previously reported [1].

Chemical Biology Screening Identifies a Vulnerability to Checkpoint Kinase Inhibitors in TSC2-Deficient Renal Angiomyolipomas.

The tuberous sclerosis complex (TSC) is a rare genetic syndrome and multisystem disease resulting in tumor formation in major organs. A molecular hallmark of TSC is a dysregulation of the mammalian target of rapamycin (mTOR) through loss-of-function mutations in either tumor suppressor TSC1 or TSC2. Here, we sought to identify drug vulnerabilities conferred by TSC2 tumor-suppressor loss through cell-based chemical biology screening. Our small-molecule chemical screens reveal a sensitivity to inhibitors of checkpoint kinase 1/2 (CHK1/2), regulators of cell cycle, and DNA damage response, in both in vitro and in vivo models of TSC2-deficient renal angiomyolipoma (RA) tumors. Further, we performed transcriptional profiling on TSC2-deficient RA cell models and discovered that these recapitulate some of the features from TSC patient kidney tumors compared to normal kidneys. Taken together, our study provides a connection between mTOR-dependent tumor growth and CHK1/2, highlighting the importance of CHK1/2 inhibition as a potential antitumor strategy in TSC2-deficient tumors.

Poly(δ-valerolactone-co-allyl-δ-valerolactone) cross-linked microparticles: Formulation, characterization and biocompatibility.

A novel polymeric material, poly(δ-valerolactone-co-allyl-δ-valerolactone) (PVL-co-PAVL), was used to manufacture microparticles (MPs) for sustained drug delivery. PVL-co-PAVL MPs were formulated using a modified oil-in-water approach, followed by a UV-initiated cross-linking process. Prepared MPs had a smooth spherical morphology and cross-linking of the copolymer was found to improve the integrity and thermal stability of the MPs. Paclitaxel (PTX) was successfully loaded into the MPs at a high drug loading capacity, using a post-loading swelling-equilibrium method. In vitro evaluation showed that the PVL-co-PAVL MPs provide sustained release of PTX, which exhibited first-order release kinetics. A subsequent pilot pharmacokinetic study was carried out on the PTX-loaded PVL-co-PAVL MPs. During this study, serum levels of PTX were monitored following subcutaneous administration of the MPs to Sprague-Dawley rats. Overall, the in vivo release of PTX from the MPs was lower than expected based on the in vitro release studies. Detectable serum levels of PTX suggest that sustained release of drug was achieved in vivo. Minimal changes in subcutaneous tissue were observed at the site of injection. Future studies will further examine the localized and systemic distribution of drug following administration in this new polymer-based MP system.

Pharmacokinetic/pharmacodynamic (PK/PD) differentiation of native and PEGylated recombinant human growth hormone (rhGH and PEG-rhGH) in the rat model of osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disease that has no FDA-approved treatment. The current standard of care does not address the regeneration of the damaged cartilage. Human growth hormone (hGH) is part of the insulin-like growth factor (IGF)-1 axis. There has been preclinical data that suggest its potential regenerative property in the joint. However, unformulated recombinant hGH (rhGH) is short-lived in the joint, and does not provide a desirable pharmacokinetic (PK) profile to support a clinical treatment paradigm. Polyethylene glycol (PEG)ylation is a potential method to extend the half-life of rhGH in the joint. The purpose of this study was to delineate the PK/PD profile of PEG-rhGH in the knee joint in a rat preclinical model of OA. After intra-articular (IA) injection of 100 microg into a rat knee joint that underwent medial meniscectomy, PEG-rhGH exhibits 2-fold longer half-lives in joint than native hGH. However, PEG-rhGH has a much longer systemic exposure. IA injections of PEG-rhGH also resulted in higher levels of IGF-1 in the joint and serum when compared with native rhGH. In order to develop PEG-rhGH as an IA therapeutic treatment for OA, careful dose selection is necessary to avoid systemic effects while retaining its anabolic efficacy in the joint.

Novel brain permeant mTORC1/2 inhibitors are as efficacious as rapamycin or everolimus in mouse models of acquired partial epilepsy and tuberous sclerosis complex.

Mechanistic target of rapamycin (mTOR) regulates cell proliferation, growth and survival, and is activated in cancer and neurological disorders, including epilepsy. The rapamycin derivative ("rapalog") everolimus, which allosterically inhibits the mTOR pathway, is approved for the treatment of partial epilepsy with spontaneous recurrent seizures (SRS) in individuals with tuberous sclerosis complex (TSC). In contrast to the efficacy in TSC, the efficacy of rapalogs on SRS in other types of epilepsy is equivocal. Furthermore, rapalogs only poorly penetrate into the brain and are associated with peripheral adverse effects, which may compromise their therapeutic efficacy. Here we compare the antiseizure efficacy of two novel, brain-permeable ATP-competitive and selective mTORC1/2 inhibitors, PQR620 and PQR626, and the selective dual pan-PI3K/mTORC1/2 inhibitor PQR530 in two mouse models of chronic epilepsy with SRS, the intrahippocampal kainate (IHK) mouse model of acquired temporal lobe epilepsy and Tsc1GFAP CKO mice, a well-characterized mouse model of epilepsy in TSC. During prolonged treatment of IHK mice with rapamycin, everolimus, PQR620, PQR626, or PQR530; only PQR620 exerted a transient antiseizure effect on SRS, at well tolerated doses whereas the other compounds were ineffective. In contrast, all of the examined compounds markedly suppressed SRS in Tsc1GFAP CKO mice during chronic treatment at well tolerated doses. Thus, against our expectation, no clear differences in antiseizure efficacy were found across the three classes of mTOR inhibitors examined in mouse models of genetic and acquired epilepsies. The main advantage of the novel 1,3,5-triazine derivatives is their excellent tolerability compared to rapalogs, which would favor their development as new therapies for TORopathies such as TSC.

Augment bone graft products compare favorably with autologous bone graft in an ovine model of lumbar interbody spine fusion.

STUDY DESIGN: This study was designed to determine whether Augment Bone Graft (Augment, Biomimetic Therapeutics, Inc., Franklin, TN) and Augment Injectable Bone Graft (Augment Injectable, Biomimetic Therapeutics, Inc., Franklin, TN), 2 combination devices comprising recombinant human platelet-derived growth factor-BB and ß-tricalcium phosphate-containing matrices, promote bone bridging in an ovine model of lumbar spine fusion. Autologous bone graft (autograft) was used as a positive control. OBJECTIVE: The purpose of this study was to determine the ability of Augment products to promote fusion of the L2-L3 and L4-L5 vertebral bodies in an ovine model. SUMMARY OF BACKGROUND DATA: In interbody spine fusion, the intervertebral disc is removed and a spacer is inserted for support and to facilitate bone growth. The fusion is commonly enhanced with grafts. Autograft is the "gold standard" but it has limitations including availability and donor-site morbidity. Synthetic graft substitutes eliminate these complications. Augment products are combination devices including recombinant human platelet-derived growth factor-BB, a well-characterized chemotactic, mitogenic, and proangiogenic protein essential in wound and bone healing. METHODS: Twenty-two sheep received an uninstrumented, double-level, interbody lumbar spinal fusion procedure using a polyetheretherketone spacer, which was either empty or packed with iliac crest autograft, Augment or Augment Injectable. The same treatment was used at both levels. Animals were 24 weeks after surgery, and fusion was assessed by micro-computed tomography (micro-CT) and histology. RESULTS: Micro-CT and histologic assessment of fusion revealed that empty controls had significantly lower fusion rates. No differences were detected among autografts, Augment, and Augment Injectable-treated specimens. Residual ß-tricalcium phosphate particles embedded in the newly formed bone were visible in Augment- and Augment Injectable-treated specimens. CONCLUSION: Augment-treated specimens had the highest fusion scores. Treatment with either of the Augment products significantly promoted interbody spine fusion compared with empty spacers and was equivalent to autograft-induced fusion. No adverse events were noted.

IGFBP-5 Metabolism Is Disrupted in the Rat Medial Meniscal Tear Model of Osteoarthritis.

Insulin-like growth factor binding protein 5 (IGFBP-5) has been proposed to promote cartilage anabolism through insulin-like growth factor (IGF-1) signaling. A proteolytic activity towards IGFBP-5 has been detected in synovial fluids from human osteoarthritic (OA) joints. The purpose of this study was to determine if protease activity towards IGFBP-5 is present in the rat medial meniscal tear (MMT) model of OA and whether inhibition of this activity would alter disease progression. Sprague-Dawley rats were subject to MMT surgery. Synovial fluid lavages were assessed for the presence of IGFBP-5 proteolytic activity. Treatment animals received intra-articular injections of vehicle or protease inhibitor peptide PB-145. Cartilage lesions were monitored by India ink staining followed by macroscopic measurement of lesion width and depth. The MMT surgery induced a proteolytic activity towards IGFPB-5 that was detectable in joint fluid. This activity was stimulated by calcium and was sensitive to serine protease inhibitors as well as peptide PB-145. Significantly, intra-articular administration of PB-145 after surgery protected cartilage from lesion development. PB-145 treatment also resulted in an increase in cartilage turnover as evidenced by increases in serum levels of procollagen type II C-propeptide (CPII) as well as synovial fluid lavage levels of collagen type II neoepitope (TIINE). IGFBP-5 metabolism is disrupted in the rat MMT model of OA, potentially contributing to cartilage degradation. Inhibition of IGFBP-5 proteolysis protected cartilage from lesion development and enhanced cartilage turnover. These data are consistent with IGFBP-5 playing a positive role in anabolic IGF signaling in cartilage.

Identification of genes responsible for osteoblast differentiation from human mesodermal progenitor cells.

Single human bone marrow-derived mesodermal progenitor cells (MPCs) differentiate into osteoblasts, chondrocytes, adipocytes, myocytes, and endothelial cells. To identify genes involved in the commitment of MPCs to osteoblasts we examined the expressed gene profile of undifferentiated MPCs and MPCs induced to the osteoblast lineage for 1-7 days by cDNA microarray analysis. As expected, growth factor, hormone, and signaling pathway genes known to be involved in osteogenesis were activated during differentiation. In addition, 41 transcription factors (TFs) were differentially expressed over time, including TFs with known roles in osteoblast differentiation and TFs not known to be involved in osteoblast differentiation. As the latter group of TFs coclustered with osteogenesis-specific TFs, they may play a role in osteoblast differentiation. When we compared the gene expression profile of MPCs induced to differentiate to chondroblasts and osteoblasts, significant differences in the nature and/or timing of gene activation were seen. These studies indicate that in vitro differentiation cultures in which MPCs are induced to one of multiple cell fates should be very useful for defining signals important for lineage-specific differentiation.