Francesca Fontana, PhD, MD
Instructor in Medicine
Internal Medicine – Cardiovascular Division
Dr. Fontana graduated in Medicine from Vita-Salute San Raffaele University (Milano, Italy), where she started working in clinical research on skeletal cancer. Transitioning to the bench, in 2012 she completed her PhD in molecular medicine, working on metabolism of multiple myeloma (MM). The following year, a fellowship from Fondazione Veronesi allowed to her to move to Washington University Schoool of medicine, where in 2014 she formally joined the laboratories of Dr. Katherine Weilbaecher and Dr. Roberto Civitelli. Her postdoctoral work revolved around the use of in vivo models to study the functions of cell adhesion molecules in bone physiology and the tumor microenvironment, or to translate characterization of myeloma metabolism of acetic acid into PET imaging.
In fall 2018, Dr. Fontana joined the “C-TRAIN” (Washington University Consortium for Translational Research in Advanced Imaging and Nanomedicine), directed by Dr. Gregory Lanza, to complement the team’s work on development of nanotherapeutics by offering a cell and cancer biology perspective. Two years later, she was appointed research instructor in medicine within the same group.
CTRAIN is an 18,000 sq. ft diversified but integrated research complex, which has been home to varying numbers of PIs since 2006. CTRAIN laboratories include formulation chemistry, nuclear magnetic resonance, molecular and cellular biology, histology, radiochemistry, small animal handling and imaging, among others. Leveraging on national and international collaborations in academia, industry, and clinic, CTRAIN aims at developing technological advances into applicable diagnostic and therapeutic tools.
Nanotherapeutics are generally characterized by a targeting group, a carrier, and a payload. The pro-drug nanomicelle system patented by the Lanza lab conjugates the drug payload and the homing ligand to phospholipids that integrate into the nanomicelle’s single-layer lipid shell, creating shelf-stable organic particles capable of packing multiple molecules of payload while remaining 60% smaller than a single IgM.
In a process named contact-facilitated drug delivery (CFDD), nanoparticles attach to their target by high affinity homing ligands, allowing the phospholipid layer to fuse with the cell membrane. Enzymatic hydrolysis then releases the active drug into the cytoplasm. A recent study describes the use of such nanoparticles designed to target drug-resistant myeloma cells (doi: 10.1158/1078-0432.CCR-20-2839)
Multiple myeloma is a relapsing-recurring neoplasia of bone marrow plasma cells, which causes systemic organ damage and severe osteolytic bone lesions. Novel treatments have significantly prolonged survival, but none of them can completely eradicate the disease: at each relapse myeloma cells accumulate resistance against drugs, while patients accumulate organ damage from off-target effects of treatments or the cancer itself. A critical need is therefore to develop treatments with extremely low toxicity but capable of overcoming drug resistance.
One of the main mechanisms that allow myeloma cells (MMC) to survive chemotherapy is the protective effect of the bone microenvironment. Integrin VLA4 mediates adhesion of MMC to the bone matrix and stromal cells, conferring resistance to the toxic effects of a wide variety of treatments. Indeed, myeloma cells surviving chemotherapy overexpress VLA4, and in vitro can be found nearly surrounded by stromal cells. Based on this observation, we hypothesized that nanotherapeutics targeting of VLA4 in conjunction with conventional chemotherapy would create a “catch-22” situation for MMC: low VLA4 expression would make them sensitive to conventional drugs, but overexpressing VLA4 to resist chemotherapy would make them vulnerable to targeted nanoparticles.
VLA4 overexpressing myelomas showed higher expression of topoisomerase 1, target of camptothecin. Matching the payload to the homing ligand, a semisynthetic camptothecin prodrug was synthetized and loaded in targeted nanomicelles. In vivo, nanoparticles distributed to a number of tissues, but payload was only transferred to VLA4-expressing cells, and preferentially to MMC surviving drug treatment. In mice with MM, combined treatment with nanoparticles and melphalan prolonged survival relative to high dose chemotherapy alone, and substantially reduced tumor burden relative to low-dose melphalan alone. No toxic effects were noted by clinical pathology, necropsy, or histology of organs sensitive to camptothecin derivatives. This suggests that nanotherapeutic targeting of drug resistant myeloma cells may be achieved with minimal to no toxicity by targeting drugs selectively to resistant cells, while lower doses of conventional drugs may be made more effective on the bulk of the tumor burden, further reducing the overall treatment-related toxicity. From a more technological standpoint, a deeper understanding of the biology of target cells and their relationship with their environment may help design more effective nanotherapeutics.
Ongoing studies aim at identifying the patient groups that would benefit the most from VLA4-targeted nanoparticles and characterize mechanisms of resistance to design corrective strategies. In collaboration with Dr. Shokeen (Radiology), theranostic implications of VLA4 binding in drug resistant cells are being addressed combining VLA4-based PET imaging and targeted treatment. In collaboration with the myeloma biobank and the Vij group, molecular patterns associated with high or low expression of VLA4 in patient cells and disease models are being investigated to find novel therapeutic targets and predictors of response in relapsing/refractory myeloma. Studies in collaboration with the Oncology Division aim at developing nanotherapeutics targeting surface proteins highly expressed in myeloma cells, which may reach a broader population of cancer cells when used as first-line agents.
Department of Orthopaedics
Dr. Shen completed his doctoral research in osteoarthritis at University of Rochester in 2012. The same year, he joined Dr. Regis O’Keefe’s laboratory as a postdoctoral researcher, and expanded his research to bone fracture repair, particularly the importance of the DNA methylation in mesenchymal stem cells. In 2018, Dr. Shen was promoted to Assistant Professor in the Department of Orthopaedic Surgery at Washington University in St. Louis. His current research interests span aspects of bone and cartilage research, and are mainly focused on injury, repair and regeneration of musculoskeletal tissues with the goal to understand the progenitor cell population, signals, and role of disease and aging on tissue injury and regeneration at the cellular and molecular level. One of the focuses in the Shen lab is to study the mechanism of epigenetic factor, DNA methyltransferase 3b in fracture nonunion under inflammatory diseases, which is a totally novel field for the bone and cartilage studies. This work has been recently funded with an R21 and two R01s.
Fracture nonunion is an exceedingly challenging clinical problem with limited and mainly invasive therapeutic interventions. It is more prevalent in patients experiencing chronic inflammatory conditions, including diabetes and rheumatoid arthritis (RA). Clinical studies have illustrated that chronic inflammation adversely affects progenitor cell differentiation and neo-angiogenesis in patients, and in turn results in the failure of bony callus formation and develops fracture nonunion. Although significant advances have been made in defining the affected cell differentiation and angiogenic process for potential pharmacologic targets, there is still an unmet clinical need for new therapeutic approaches for fracture repair and nonunion treatment, especially for older patients those with comorbidities such as diabetes and RA. Indeed, serum transfer-induced RA (K/BxN) mice displayed fracture nonunion with absence of fracture callus, diminished angiogenesis and fibrotic scar tissue formation (Fig. 1), leading to failure of biomechanical properties, which represented the major manifestations of atrophic nonunion in clinic. However, a gap of knowledge remains regarding the downstream targets of this phenomenon.
To bridge this gap, we have focused on epigenetics, especially DNA methylation, which has emerged as a critical modulator in various cells, under inflammatory condition such as RA. Indeed, recent epigenome studies from fracture patients revealed differential methylation loci in human cells, suggesting that DNA methylation is involved in fracture repair process. Importantly, our preliminary data show that the epigenetic enzyme Dnmt3b is highly expressed in fracture callus during fracture repair and Dnmt3b is the major DNA methyltransferase (Dnmt) responsive to cytokines in progenitor cells and chondrocytes. Mechanistically, 1) progenitor cell differentiation defect mediated by inflammation and Dnmt3b LOF coincide with upregulation of Rbpjx in progenitor cells and Rbpjx inhibition can restore differentiation capacity in vitro; 2) angiogenesis defect mediated by inflammation and Dnmt3b LOF coincide with downregulation of OPN (Osteopontin) and CXCL12 (C-X-C Motif Chemokine Ligand 12) and exogenous OPN and CXCL12 can restore angiogenesis capacity in vitro. In collaboration with Dr. Jianjun Guan, we propose to develop potentially therapeutical approaches for fracture nonunion treatment via delivery of growth factors (OPN and CXCL12) and genetically modified progenitor cells (① feedback-controlled anti-inflammatory stem cells with Dnmt3b overexpression and ② stem cells with locally DNA methylation modified Rbpjx into fracture site by PCL scaffold.
Additional information about the lab is available on our website:
Department of Pathology and Anatomical Sciences
University of Missouri School of Medicine
Dr. Middleton joined the Department of Pathology & Anatomical Sciences in the University of Missouri School of Medicine in 2012. Originally trained as a paleontologist and functional anatomist, he completed his doctoral research at Brown University avian foot evolution. He remained at Brown as a postdoctoral research associate, studying the aerodynamics of bat flight and the mechanical properties of bat wing bones. An NIH NRSA Postdoctoral Research fellowship at the University of California, Riverside and Brown motivated a switch to using mouse models to study the genetic and plastic responses to selection for high levels of voluntary activity on the musculoskeletal system. Past projects in the lab have included penguin evolution, the effects of atmospheric oxygen on skeletal ontogeny in alligators, and the evolution and biomechanics of bird heads. Recently, we began developing non-linear Bayesian models to estimate growth trajectories in the human head. Despite these varied interests, the main focus of the lab is the interplay between genetic background and lifetime voluntary activity in determining skeletal structure & function.
We study mice that have been artificially selected for high levels of voluntary wheel activity as a model organism to explore the diverse effects of high levels of voluntary exercise on musculoskeletal ontogeny, form, and function. For over eighty generations, these mice have been bred for increased voluntary activity and run nearly three times farther than randomly bred controls, often more than 25 km in one night. Such repetitive loading of the skeletal system provides a unique opportunity to study the interaction between genetic background and activity on bone growth, form, and function. Work has focused on the response of the skeletal system to both long-term selection and acute exercise. Initial studies focused on morphological changes associated with either artificial selection or exercise, or the combined effects of the two, with the goal of addressing fundamental questions about the skeletal response to exercise and changes to the underlying genetic landscape. Several studies using both traditional morphometric techniques and fine-scale micro-computed tomography (µCT) revealed significant skeletal differences between selected and control mice. Two recent projects have been enhanced by working with the Musculoskeletal Research Center’s Structure and Strength Core.
In the first of these projects, we compared pelvis and hind limb shapes between randomly bred control mice and those artificially selected for high activity (Figure 1).
In the pelvis and proximal femur, we find many areas of significant morphological divergence, which are associated with altered gait kinematics and kinetics in selected lines. There appears to be a proximodistal gradient, with change concentrated in the pelvis and hip, with fewer differences observed in the tibia. In a second project, we compared the evolved cranial morphology in exercise-selected mice to that of controls, which shows distinct patterns of change in the neurocranium vs. the viscerocranium (Figure 2).
Exercise selected mice show significantly expanded neurocranium and posterior skull, with wider nuchal plane and foramen magnum. In contrast, these mice have significantly reduced viscerocrania, including a smaller overall face and shorter, narrower palate. In additional to these evolved responses, both lines show unique patterns of plastic responses to exercise. Future research will investigate the ontogenetic patterns and mechanistic basis for these differences.
Additional information about the lab is available on our website: https://www.middletonlab.org/
Assistant Professor of Medicine Division of Bone & Mineral Diseases
Dr. Craft completed her graduate work in cancer drug discovery at Northwestern University’s Feinberg School of Medicine in 2007. The same year, she joined the Department of Cell Biology and Physiology at Washington University as a postdoctoral fellow in the laboratory of Dr. Bob Mecham. During her fellowship, Dr. Craft investigated extracellular matrix (ECM)-mediated regulation of TGFß within the skeleton. In 2012, Dr. Craft was promoted to Assistant Professor in the Department of Cell Biology and Physiology. Early in her independent career, Dr. Craft was funded by the American Diabetes Association to define the role of the ECM in adiposity and metabolic disease. During this time, she found that loss of a single ECM protein, MAGP1, was sufficient to predispose mice to metabolic syndrome (obesity and diabetes), pathologic bone marrow adipose tissue (BMAT) expansion, and bone fractures. The finding that diabetes was linked to bone fragility and altered BMAT adipocyte function led to a collaboration with Dr. Erica Scheller. In response to the success of their collaboration, in 2016 Dr. Craft transferred to the Division of Bone and Mineral Diseases to create a novel joint laboratory with Dr. Scheller which investigates the relationship between nerves and bone, with current emphasis on neuropathy and skeletal metabolism in diabetes. The lab’s research in bone marrow adiposity (BMA), diabetic neuropathy, and skeletal ECM were recently highlighted through oral presentations at the 2018 BMA Society meeting in Lille, France and the 2018 ASBMR meeting in Montreal, Canada. In her spare time, Dr. Craft enjoys spending time outdoors with her husband and children at their farm in Sullivan, Missouri.
For more information about Dr. Craft’s research interests, please visit the lab’s website:
Assistant Professor of Orthopaedic Surgery
Hand & Microvascular Surgery Service
The Orthopaedic Nerve Research Lab is a collaborative effort between Dr. Christopher Dy and Dr. David Brogan. Over the past two years, the laboratory has been focused on translational and epidemiologic research regarding complex peripheral nerve injuries. Dr. Dy has received funding from the NIH to explore the utilization of qualitative research methods in describing the psychosocial effects of brachial plexus injuries on patients. His previous work has also focused on understanding the epidemiology of these severe injuries to better understand injury patterns.
Dr. Brogan’s focus is on translational research to better improve the diagnosis of nerve injuries in the operating room. He has received funding from the American Foundation for Surgery of the Hand to evaluate the application of laser Doppler blood flow technology through the diagnosis of peripheral nerve crush injuries. The lab also works closely with Dr. Samuel Achilefu in the Optical Radiology Lab. The current work explores the utilization of near-infrared optical probes to better understand the cascade and sequence of events that occur surrounding a peripheral nerve injury. Our hope is to eventually develop tools that can be utilized with existing equipment in the operating room to assist with image-guided nerve surgery. The lab also has ongoing collaborations with the Department of Genetics as well as the Department of Neurosurgery. Dr. Dy and Dr. Brogan are committed to continuing their work to hopefully improve the diagnosis of nerve injuries, as well as mechanisms for better surgical treatment and the aftercare of patients affected by these life-altering injuries.
Research Associate Professor of Medicine
Division of Geriatrics & Nutritional Science
The Finck Lab is interested in understanding the regulation of intermediary metabolism in a variety of tissues, including skeletal muscle. For many years, we have been interested in the lipin family of proteins (lipin 1, 2, and 3). Lipins are intracellular proteins that primarily act as lipid phosphatase enzymes in the pathway of triglyceride synthesis. Interestingly, these proteins can also translocate to the nucleus to directly regulate gene expression by interacting with DNA-bound transcription factors that control the expression of metabolic enzymes. Thus, lipin proteins can control metabolism at multiple regulatory levels by affecting gene expression and by their effects as phosphatase enzymes.
Lipin 1 is the lipin isoform that is most highly expressed in skeletal muscle. Recent work has demonstrated that rare mutations in the gene encoding lipin 1 in humans (LPIN1) lead to recurrent, episodic rhabdomyolysis that manifests mainly when these patients are children. In collaboration with Dr. Robert Bucelli, who has identified patients with LPIN1 mutations in the St. Louis area, we characterized a novel mutation in lipin 1 and teased apart the effects of several disease-causing mutations on the activities of the protein. While many mutations were complete loss of function, it was determined that some of the mutations caused a loss of enzymatic activity of lipin 1 protein without affecting its ability to regulate gene transcription. This suggests that defects in lipin’s lipid phosphatase activity leads to pathologic changes that explain the etiology of rhabdomyolysis in these patients.
To better understand this, Dr. George Schweitzer in the Finck group created novel mice with skeletal muscle-specific knockout of lipin 1. While outwardly normal, skeletal muscle of the knockout mice exhibited a chronic myopathy with ongoing muscle fiber necrosis and regeneration. These mice also exhibited accumulation of the lipid substrate of lipin 1, phosphatidic acid, in muscle. Additionally, lipin 1-deficient mice had abundant, yet abnormal mitochondria likely due to impaired autophagy. Phosphatidic acid suppresses autophagy by a variety of mechanisms, including by stimulating the activity of the mTOR kinase signaling cascade, which can be targeted by pharmacologic means.
While these mice do not completely phenocopy the episodic nature of patients with the human LPIN1 mutation, these data suggest that mice lacking lipin 1-mediated PAP activity in skeletal muscle may serve as a model for further exploration of the mechanisms by which lipin 1 deficiency leads to myocyte injury. We also hope to use these mice to test potential therapeutic or preventative approaches. Lastly, the lab is also interested in determining whether lipin 1 deactivation in other more common forms of muscular dystrophy may contribute to the progression of those diseases by similar mechanisms.
Department of Mechanical Engineering and Materials Science
The Polymers, Interfaces, and Regenerative Medicine lab led by Prof. Jianjun Guan at the Department of Mechanical Engineering and Materials Science is interested in biomimetic biomaterials synthesis and scaffold fabrication; bioinspired modification of biomaterials; injectable and highly flexible hydrogels; bioimageable polymers for MRI and EPR imaging and oxygen sensing; mathematical modeling of scaffold structural and mechanical properties; stem cell transplantation; and bone, skeletal muscle, skin, and cardiac regeneration.
One of the projects in Dr. Guan’s lab is to regenerate vasculature and skeletal muscle in ischemic limbs. Critical limb ischemia (CLI) is a severe peripheral artery disease with high rates of limb loss and mortality. It is featured by low blood perfusion, extensive tissue ischemia, and degenerated skeletal muscle. Quick vascularization to restore blood perfusion, and fast muscle regeneration to restore normal function, represent the optimal goals for CLI treatment. Currently there is no efficient treatment available, although stem cell therapy is one of the most promising strategies. However, current stem cell therapy experiences low efficacy largely due to inferior cell survival and paracrine effects under the extremely low oxygen condition of ischemic limbs. Dr. Guan’s lab is developing a new cell delivery system that continuously releases appropriate concentration of oxygen to simultaneously improve stem cell survival and paracrine effects, resulting in quick vascularization and muscle regeneration. Paracrine effects concurrently provide multiple growth factors critical for limb regeneration, which cannot be readily achieved by growth factor therapy. The team found that the oxygen-releasing cell delivery system can fully restore blood perfusion and muscle contractility in four weeks.
Department of Orthopaedic Surgery
Dr. Pascual-Garrido, MD, PhD, joined Washington University in 2017. She was recruited as an assistant professor in the area of Hip Joint Preservation and Adult Reconstruction. Originally from Argentina, she came to the United States to work in the laboratory of Dr. Susan Chubinskaya at Rush University Medical Center in Chicago. It was at this point that Dr. Pascual-Garrido established an interest in joint pathology and a desire to better understand the factors leading to the development of OA. Her research development has been complemented by outstanding clinical training. Dr. Pascual Garrido completed 3 years of fellowship training in the area of sports medicine and joint arthroplasty. One of these years was spent at the Hospital of Special Surgery, NY, with Dr. Scott Rodeo, an experience that further stimulated Dr. Pascual Garrido to develop a career as a clinician scientist. She has established a busy practice at Washington University, focusing mainly in the treatment of the pre-arthritic hip. Additionally, she has started a lab that is trying to identify critical biologic events that are mediators of the OA cascade in hip Femoroacetabular impingement (FAI). Current reports indicate an etiologic role of FAI in up to 50% of hip OA cases. Recently, her lab found for the first time a specific location of inflammatory and catabolic markers during disease progression in hip FAI, providing novel evidence that the impingement zone of hips with FAI show an osteoarthritic phenotype with degeneration and markedly elevated levels of selected inflammatory and catabolic, suggesting that the chondrocytes are metabolically active. Additionally, transcriptome analysis from patients with FAI and advanced OA suggest different enriched biological processes with specific activation pathways at early stage of hip OA disease. Currently, her lab continues to work on identifying a catabolic state in articular chondrocytes from the impingement zone in hip FAI and characterize spectrum of disease through gene expression and DNA methylation or epigenetic changes. Dr. Pascual believes that integrating epigenomic and transcriptome data will allow a better understanding of how the identified loci may contribute to OA pathogenesis. Finally, her lab is characterizing a small animal model of hip FAI and secondary OA. This animal model could provide a novel opportunity to test future interventional studies for the treatment of hip OA. Through established collaboration with musculoskeletal researchers at the MRC and clinician scientists at Wash U Orthopedics, there is true a potential to uncover for the first time the early pathological pathways associated with hip OA secondary to hip FAI. She hopes to make significant and meaningful contributions in the care of young adults with pre-OA hip disorders, through the process of basic scientific discovery and its translation to patient care.
Schema of the proposed structural changes in the developing osteoarthritis secondary to FAI. ADAMTS-4: a disintegrin and metalloproteinase with thrombospondin motif-4, COL2: type II collagen, DDH: developmental dysplasia of the hip, ECM: extracellular matrix, FAI: femoroacetabular impingement, IL-1β: interleukin-1 beta, MMP-13: matrix metalloproteinase-13, NITEGE: aggrecan monoclonal antibody to C-terminal neoepitope, OA: osteoarthritis. Immunohistochemistry of the cartilage from head-neck area in corresponding groups. The arrows show the zone of immunopositive cells, and the dotted line shows tidemark on each panel. Scale bar = 500μm. (B) Bar Graphs are represent number of IL-1β, MMP-13, ADAMTS-4, COL2, and NITEGE positive cells across groups. Statistical differences were observed among groups (* shows the significant differences compared to the control, p<0.05).
Director – Division of Rheumatology Professor of Medicine and Pathology and Immunology
One of the focuses in the Pham lab is to develop novel approaches to deliver therapeutics that will halt or reverse joint inflammation and degeneration in preclinical models of rheumatoid arthritis and osteoarthritis, with the ultimate goal of translating these findings to the clinic. These projects represent a team-science, interdisciplinary approach to arthritis research, combining the Pham lab expertise in basic mechanisms underpinning these rheumatic conditions with innovative bioengineering advances in nanomedicine and regenerative medicine pioneered by outstanding collaborators in the Departments of Orthopaedics and Bioengineering. We collaborated with Drs. Linda Sandell, Farooq Rai, Farshid Guilak, and Sam Wickline (currently at University of South Florida) to deliver a peptide-siRNA nanocomplex targeting the NF-kB pathway to mitigate inflammation in murine models of rheumatoid arthritis and post-traumatic osteoarthritis. We have shown that peptide- NF-kB p65 siRNA nanocomplex suppresses experimental rheumatoid arthritis. We also leveraged the expertise and support of the Musculoskeletal Research Center, specifically the Structure and Strength Core and the Musculoskeletal Histology and Morphometry Core to conduct a murine model of controlled knee joint impact injury to test the hypothesis that delivery of peptide- NF-kB p65 siRNA nanocomplex in the immediate aftermath of joint injury will prevent cartilage degeneration and the eventual development of post-traumatic osteoarthritis. We showed that peptide-siRNA nanocomplex suppresses NF-kB activation (Figure 1) and mitigates several important early events post injury, including chondrocyte apoptosis, thus reducing the extent of cartilage injury and reactive synovitis. In addition to structural changes, we have now shown that treatment with peptide-siRNA nanocomplex is associated with improvement in pain sensitivity post injury. These findings may lead to the development of a first-in-class disease-modifying nanotherapeutic approach to prevent post-traumatic osteoarthritis. More recently we have also collaborated with Dr. Guilak and Drs. Yun-Rak Choi and Kelsey Collins (postdoctoral scholars in the Guilak lab) to test the ability of a tissue-engineered stem cell-based system with autoregulated cytokine antagonist delivery to mitigate inflammation in a robust murine model of rheumatoid arthritis. The system employs genome-engineered pre-differentiated iPSCs to deliver anti-cytokine therapeutics, the production of which is driven by endogenous levels of inflammatory cytokines. Our data suggest that this “SMART” cell-based delivery of IL-1 receptor antagonist suppresses inflammation, prevents bone erosions and mitigates pain induced by inflammatory arthritis. In addition to work in pre-clinical models of diseases, our lab is also actively involved in several translational projects. Our translational work is partially supported by the Washington University Rheumatic Diseases Research Resource-based Center (WU-RDRRC), a NIAMS-funded mechanism. WU-RDRRC’s mission is to promote cross-disciplinary collaborations and to stimulate the development of new initiatives that will advance the pace of discovery, with the goal of disseminating and implementing research findings into the practice of personalized medicine.