PI:Paolo Bonato, PhD
Abstract:Comparing a Passive-Elastic and a Powered Prosthesis in Transtibial Amputees with Diabetes Mellitus
Many North Americans are currently living with a limb loss. In 2005, there were 1.6 million lower-limb amputees in the United States, and it is estimated that the prevalence will rise to 3.6 million in 2050. About 58% of all lower-limb amputations result from diabetes and its complications. Amputees with diabetes are typically older adults (i.e. 65+ years of age) and suffer from added co-morbidities. Being an amputee because of diabetes complications is associated with an augmented risk (compared to traumatic amputees) of cardiovascular death, non-fatal myocardial infarction, stroke, subsequent amputations and interventions for peripheral vascular diseases (peripheral angioplasty, stent or bypass surgery). Secondary prevention and risk reduction strategies in amputees with diabetes mellitus have been developed and are aimed at controlling risk factors. A commonly proposed intervention is physical activity. Aerobic exercise alone has been demonstrated to reduce cardiovascular complications and overall mortality, improve glycemic indices and weight control, and has been associated with an improved quality of life in diabetic patients. One of the most accessible physical activities for every individual is locomotion, which in amputees requires the use of a prosthesis with or without other assistive devices.
Transtibial amputees expend approximately 38% more energy during level-ground walking than non-amputees. The increased metabolic cost of ambulation limits the duration and speed of walking, often resulting in a sedentary life-style. Adopting a physically-active routine in accordance with secondary prevention recommendations is challenging for amputees. Powered ankle-foot prostheses generating net positive work during the terminal portion of the stance phase of gait were recently developed and promise to offer better gait biomechanics. We hypothesize that transtibial amputees due to diabetes mellitus complications who use a powered ankle-foot prosthesis could achieve better gait biomechanics at a lower metabolic cost than when using a passive-elastic foot prosthesis. We also hypothesize that the improved gait biomechanics and related improvements in energy efficiency of ambulation would result in an enhanced activity level in diabetic patients who suffered a transtibial amputation.
To test these hypotheses, we propose to perform laboratory and field (i.e. in the home and community settings) tests with both powered and passive-elastic ankle-foot prostheses. Biomechanical tests and oxygen consumption measurements will be performed in the Motion Analysis Laboratory at Spaulding Rehabilitation Hospital. In addition, data will be gathered in amputees when using powered and passive-elastic ankle-foot prostheses using monitoring technology designed to capture activity level in the home and community settings. We anticipate that the hypothesized biomechanical benefits of powered prostheses would encourage diabetic patients who suffered the amputation of a lower limb to make a transition from a sedentary to a more physically-active lifestyle, hence improving health status and quality of life. As part of Spaulding Rehabilitation Hospital, the Motion Analysis Laboratory is committed to enhancing mobility in those suffering mobility-limiting conditions such as an amputation. With the proposed research, we would pursue a path toward clinical trials that could bring new technologies to diabetic patients in order to transform their lives.
PI: Mary Bouxsein, PhD
Abstract: Skeletal Fragility in Type 2 Diabetes Mellitus
Recent studies have shown that individuals with type 2 diabetes mellitus (T2DM) have increased fracture risk, despite being obese and having normal to high bone mineral density, both of which are presumed to protect against fractures(1). The public health impact of skeletal fragility in this group will only grow due to the aging of the population and the significantly increasing prevalence of diabetes. Over the next fifty years the largest increase in diabetes will be in those aged 75 years and greater, with increases projected as high as 336% for this oldest age group (2) .
To date, there is a very poor understanding of the mechanisms underlying skeletal fragility in adults with T2DM. Several mechanisms have been proposed to contribute to the increased fracture risk seen in individuals with T2DM, including an increased propensity to fall, altered bone microarchitecture, and poor bone quality, due to an accumulation of advanced glycation endproducts (AGE) that decrease bone strength (3). In particular, in vitro incubation of bone with ribose has confirmed that AGE accumulation negatively impacts bone biomechanical properties (4,5). However, no studies have investigated bone samples from patients with diabetes to determine whether AGE content is higher and bone mechanical properties are deteriorated. We propose to determine the contribution of AGE accumulation to altered bone biomechanical properties by studying femoral neck bone specimens retrieved from T2DM and control patients (n=20/group) undergoing total hip replacement. We will measure biomechanical properties of the bone tissue using reference point indentation, a novel technique for directly assessing the mechanical behavior of the bone matrix (6,7). After mechanical testing, proteins will be extracted from bone specimens and used to measure collagen and AGEs content (8). In addition, we will collect blood from each patient in order to relate serum pentosidine, insulin and HbA1c to levels of AGE in the bone itself. Access to the patients and bone specimens is made feasible by colleagues in the Department of Orthopedic Surgery. Results from this study will provide novel information on the potential contribution of poor bone quality to skeletal fragility in patients with T2DM.
- Schwartz AV, Sellmeyer DE 2007 Diabetes, fracture, and bone fragility. Curr Osteoporos Rep 5(3):105-11.
- Boyle JP, Honeycutt AA, Narayan KM, Hoerger TJ, Geiss LS, Chen H, Thompson TJ 2001 Projection of diabetes burden through 2050: impact of changing demography and disease prevalence in the U.S. Diabetes Care 24(11):1936-40.
- Hamann C, Kirschner S, Gunther KP, Hofbauer LC 2012 Bone, sweet bone--osteoporotic fractures in diabetes mellitus. Nat Rev Endocrinol 8(5):297-305.
- Vashishth D, Gibson GJ, Khoury JI, Schaffler MB, Kimura J, Fyhrie DP 2001 Influence of nonenzymatic glycation on biomechanical properties of cortical bone. Bone 28(2):195-201.
- Tang SY, Zeenath U, Vashishth D 2007 Effects of non-enzymatic glycation on cancellous bone fragility. Bone 40(4):1144-51.
- Hansma P, Yu H, Schultz D, Rodriguez A, Yurtsev EA, Orr J, Tang S, Miller J, Wallace J, Zok F, Li C, Souza R, Proctor A, Brimer D, Nogues-Solan X, Mellbovsky L, Pena MJ, Diez-Ferrer O, Mathews P, Randall C, Kuo A, Chen C, Peters M, Kohn D, Buckley J, Li X, Pruitt L, Diez-Perez A, Alliston T, Weaver V, Lotz J 2009 The tissue diagnostic instrument. Rev Sci Instrum 80(5):054303.
- Diez-Perez A, Guerri R, Nogues X, Caceres E, Pena MJ, Mellibovsky L, Randall C, Bridges D, Weaver JC, Proctor A, Brimer D, Koester KJ, Ritchie RO, Hansma PK 2010 Microindentation for in vivo measurement of bone tissue mechanical properties in humans. J Bone Miner Res 25(8):1877-85.
- Sroga GE, Karim L, Colon W, Vashishth D 2011 Biochemical characterization of major bone-matrix proteins using nanoscale-size bone samples and proteomics methodology. Mol Cell Proteomics 10(9):M110 006718.
PI: Mark A. Herman, MD
The metabolic syndrome is a cluster of disorders that includes obesity, hypertriglyceridemia, hypertension, and insulin resistance which predispose to the development of diabetes and cardiovascular disease. Emerging epidemiological and clinical data indicate that increased sugar, and particularly fructose, ingestion is a major contributor to the development of metabolic syndrome. Despite intensive investigation over the last three decades, the molecular mechanisms underlying the development of the metabolic syndrome and specifically the mechanisms by which high-fructose feeding cause metabolic dysfunction remain elusive. Carbohydrate Responsive-Element Binding Protein (ChREBP) is a transcription factor which is activated by products of carbohydrate metabolism. It is a master regulator of glycolytic and lipogenic genetic programs. We have recently discovered a novel, potent isoform of ChREBP, ChREBP-beta. Our recent data demonstrates that expression of hepatic ChREBP-beta and its targets are robustly increased by fructose ingestion, but only modestly increased by glucose ingestion. Mice with whole-body genetic deletion of both ChREBP isoforms are intolerant to dietary fructose and die within several days of high-fructose feeding. Thus, ChREBP is essential for a normal metabolic response to dietary fructose. Here, we propose that hepatic ChREBP is a key fructose sensor and that with excessive fructose ingestion, hepatic ChREBP plays a critical role in the development of the metabolic syndrome. Our aim in this proposal is to begin to investigate the hypothesis that hepatic ChREBP, and specifically ChREBP-beta, is a critical regulatory element which responds to fructose ingestion and mediates a regulatory response that underlies development of diverse features of the metabolic syndrome. These studies will provide novel insights into the mechanisms of nutrient induced metabolic syndrome.
PI:Philip Liu, PhD
Abstract: MRI of Pericyte Reduction to Quantitate Diabetic Retinopathy in vivo
Diabetic retinopathy is a common and specific microvascular complication of diabetes. The ability to diabetic retinopathy in inaccessible retina would benefit both our understanding of diabetic retinal degeneration and our ability to develop appropriate therapies. We have developed a cell targeting MR contrast agent that links contrast agents with small phosphorothioate-modified antisense DNA (sODN) of 18-26 nucleotides in length to enable in vivo reporting and quantitatively charaterizing pericyte in a mouse model of cerebral ishcemia with a leaky blood brain barrier (BBB) by magnetic resonance imaging (MRI). The specificity of this novel contrast agent is mediated by retention of sODN bound to mRNA target but exclusion of free or non-targeting sODN in all living cells including multipotent PC12 cells, fresh brain slices and living mouse brains. These sODN, when labeled with superparamagnetic iron oxide nanopaticels (SPION) via NeutrAvidin-biotin linakage, can be made as multimodal for MRI in vivo and electronic microscopy (EM) ex vivo. We have several SPION-sODNs that co-register cohorts of cells with a resolution of < 0.03 mm3. These SPION-sODNs detect a wide range of physiologic and pathologic processes, such as gliogenesis, angiogenesis and leaky BBB in progress, including living C57black6 mice with compromised BBB after forebrain ischemia using bilateral carotid occlusion (BCAO). We have demonstrated delivery of our contrast agent as eye drops (Liu et al., FASEB J 22:1193, 2008). Recently, we show caveolae mediates the uptake of SPION-sODN after non-invasive intraperitoneal (i.p.) injection; SPION-sODN reports mRNA expression with a positive correlation (R2 = 1.0, p = 0.02) between the elevation in the rate of MR signal reduction (DR2*) value above the baseline (in vivo) and mRNA copy number (ex vivo TaqMan analysis. Our hypothesis is that the retention of pericyte-targeting contrast agent can be quantitatively measure in vivo with longitudinal MR in diabetic models. If successfully demonstrated, SPION-sODN will enable specialized cells targeting by MRI in clinical research. We will:
Aim 1: Demonstrate effective transfection of pericyte-targeting MR contrast agents to the retina of mouse diabetic models. Because the eye has no BBB but has blood-retinal barrier which becomes leaky with the progression of diabetes, we will test if SPION-actin will be delivered to the retina in diabetic mouse model of type 1 (C57BL/6 mice with streptozotocin-induced diabetes) and type 2 diabetes (db/db mice) and age-matched and sex-matched controls. We aim to demonstrate that SPION-actin effectively transfect the retina (in vivo). The controls are sODN-gfap and -Ran. We will compare the uptake and retention of Cy5.5-SPION-sODN. We will deliver pericyte-targeting MR contrast agent to the retina in mouse diabetic models (type I and type II). We will first apply iron oxide based agent because iron oxide is biocompatible and we observed no toxicity in more than 2000 mice that we have worked with. If the background noise is high, we will use gadolinium in the place of iron oxide, a T1 contrast agent. We will optimize the signal of MR-visible probes to demonstrate that, at the appropriate dose, SPION- actin will generate MR signal (DR2* maps) with optimal signal to noise ratio from Actin-expressing cells.
Future direction: Although we have focused on neuroscience of stroke and drug addiction, we have received supports from various agencies: the NIH (3R21, 4R01 and one EUREKA from NINDS, NIBIB, NIDA, NCAM), the National Science Foundation, and the American Heart Association (3 GIA and one EIG) for the development of this technology. Specific detection of pericyte reduction in the retina may allow quantitative measurement of reduced retinal regeneration. When this pilot project is successfully demonstrated for its feasibility, we are in a position to submit R01, P01 or BRP-type R01. We will propose to validate correlation of SPION-sODN between ROI of in vivo MRI and ex vivo histology. We will focus on (1) uptake and distribution of SPION-actin and SPION-nestin by in vivo MRI and ex vivo transmission EM (2) the correlation between DR2* value and mRNA copy number by PCR, with and without treatments.
PI:Klaus vanLeyen, PhD
Abstract: Can 12/15-Lipoxygenase Inhibition Protect Against Increased Brain Injury After Experimental Stroke in Diabetic Animals?
Abstract – In addition to conferring a higher risk of stroke and cardiovascular events, diabetes mellitus also leads to increased severity of stroke and worse outcome. The reasons for this are not entirely clear, but relate to deficiencies in the brain vasculature of diabetics. Our lab has previously shown that the enzyme 12/15-lipoxygenase (12/15-LOX) is a major contributor to both neuronal cell death, as well as vascular injury and edema formation following experimental stroke in mice (van Leyen et al., Stroke 2006; Jin et al., Stroke 2008). Using the db/db mouse as a model for type 2 diabetes, we have now generated Preliminary Data to show that 12/15-LOX is increased on the ischemic side of the brain in a stroke model of distal middle cerebral artery occlusion, in what appears to be vascular cells. Our Central Hypothesis states that 12/15-LOX, which is known to be up-regulated in diabetes, is a major contributor to vascular failure and increased brain injury following stroke in diabetic animals.
We plan to investigate this hypothesis in the following Specific Aims:
- Investigate 12/15-LOX expression over time in db/db mice subjected to transient focal ischemia, and compare to heterozygous and matched wild-type mice. We expect to find not just increased baseline levels of 12/15-LOX, but also sharper increases in the ischemic brain of the homozygous db/db mice. We also expect increased infarct sizes and vascular permeability in the db/db mice. 12/15-LOX expression will be detected by immuno-histochemistry, and colocalization with cell-type specific marker proteins will be used to determine which cells express 12/15-LOX. Infarct sizes will be determined by TTC staining, and vascular permeability will be detected as leakage of Evans Blue dye.
- Study 12/15-LOX inhibition as treatment option in db/db mice subjected to focal ischemia. We have recently introduced a novel inhibitor of 12/15-LOX, LOXBlock-1, which blocks 12/15-LOX enzyme activity in vivo (Yigitkanli et al., Annals of Neurology in Press). We will administer varying doses of LOXBlock-1 by intraperitoneal injection, and measure infarct sizes and vascular leakage as outcome measures. We expect to find an optimal dose for protection of the diabetic mice against stroke-induced injury.
At the end of this Pilot Study, we should have a clear picture of the damaging effects of increased 12/15-LOX in the diabetic brain, along with possible therapeutic benefits of 12/15-LOX inhibition. This may be important not just for the treatment of stroke in diabetic patients, but also for other diabetes-related complications where 12/15-LOX contributes to disease severity.