Faculty Bibliography

OBJECTIVE:One driving factor in the progression to posttraumatic osteoarthritis (PTOA) is the perpetuation of the inflammatory response to injury into chronic inflammation. Molecular imaging offers many opportunities to complement the sensitivity of current imaging modalities with molecular specificity. The goal of this study was to develop and characterize agents to image hyaluronan (HA)-mediated inflammatory signaling. DESIGN/METHODS:We developed optical (Cy5.5-P15-1) and magnetic resonance contrast agents (Gd-DOTA-P15-1) based in a hyaluronan-binding peptide (P15-1) that has shown anti-inflammatory effects on human chondrocytes, and validated them in vitro and in vivo in two animal models of PTOA. RESULTS:In vitro studies with a near infrared (NIR) Cy5.5-P15-1 imaging agent showed a fast and stable localization of Cy5.5-P15-1 on chondrocytes, but not in synovial cells. In vivo NIR showed significantly higher retention of imaging agent in PTOA knees between 12 and 72h (n=8, Cohen's d>2 after 24h). NIR fluorescence accumulation correlated with histologic severity in cartilage and meniscus (ρ between 0.37 and 0.57, p<0.001). By using in vivo magnetic resonance imaging with a Gd-DOTA-P15-1 contrast agent in 12 rats, we detected a significant decrease of T1 on injured knees in all cartilage plates at 48h (-15%, 95%-confidence interval (CI)=[-18%,-11%] []) while no change was observed in the controls (-2%, 95%-CI=[-5%,+1%]). CONCLUSIONS:This study provides the first in vivo evidence that hyaluronan-related inflammatory response in cartilage after injury is a common finding. Beyond P15-1, we have demonstrated that molecular imaging can provide a versatile technology to investigate and phenotype PTOA pathogenesis, as well as study therapeutic interventions.

Our aim was to test the effectiveness of ultrasound-guided intra-articular (IA) injection into the knee joint of rodents by an inexperienced operator compared with standard landmark-guided IA injections by a trained injector. Fifty landmark-guided and 46 ultrasound-guided IA injections in 49 rats were analyzed. Animal positioning and injection protocol were designed for use with the ultrasound system. Injection delivery was verified with a secondary imaging modality. We compared the success of IA injections by method (landmark and ultrasound-guided), adjusting for all other confounding factors (age, weight, experience, laterality and presence of surgery). Ultrasound-guided injections had higher success rates overall (89% vs. 58%) and helped to reduce the number of failed attempts per injection. None of the cofounding factors influenced the success of injection. In conclusion, we found higher accuracy for ultrasound-guided IA injection delivery than the traditional landmark-based injection method and also the technical feasibility for untrained personnel.

OBJECTIVE:To validate a radial imaging spin-echo diffusion tensor (RAISED) sequence for high-resolution diffusion tensor imaging (DTI) of articular cartilage at 3 T. METHODS:The RAISED sequence implementation is described, including the used non-linear motion correction algorithm. The robustness to eddy currents was tested on phantoms, and accuracy of measurement was assessed with measurements of temperature-dependent diffusion of free water. Motion correction was validated by comparing RAISED with single-shot diffusion-weighted echo-planar imaging (EPI) measures. DTI was acquired in asymptomatic subjects (n = 6) and subjects with doubtful (Kellgren-Lawrence [KL] grade 1, n = 9) and mild (KL = 2, n = 9) symptomatic knee osteoarthritis (OA). MD and FA values without correction, and after all corrections, were calculated. A test-retest evaluation of the DTI acquisition on three asymptomatic and three OA subjects was also performed. RESULTS:The root mean squared coefficient of variation of the global test-restest reproducibility was 3.54% for MD and 5.34% for FA. MD was significantly increased in both femoral condyles (7-9%) of KL 1 and in the medial (11-17%) and lateral (10-12%) compartments of KL 2 subjects. Averaged FA presented a trend of lower values with increasing KL grade, which was significant for the medial femoral condyle (-11%) of KL 1 and all three compartments in KL 2 subjects (-18 to -11%). Group differences in MD and FA were only significant after motion correction. CONCLUSION/CONCLUSIONS:The RAISED sequence with the proposed reconstruction framework provides reproducible assessment of DTI parameters in vivo at 3 T and potentially the early stages of the disease in large regions of interest. KEY POINTS/CONCLUSIONS:• DTI of articular cartilage is feasible at 3T with a multi-shot RAISED sequence with non-linear motion correction. • RAISED sequence allows estimation of the diffusion indices MD and FA with test-retest errors below 4% (MD) and 6% (FA). • RAISED-based measurement of DTI of articular cartilage with non-linear motion correction holds potential to differentiate healthy from OA subjects.

PURPOSE: Diffusion tensor imaging (DTI) of articular cartilage is a promising technique for the early diagnosis of osteoarthritis (OA). However, in vivo diffusion tensor (DT) measurements suffer from low signal-to-noise ratio (SNR) that can result in bias when estimating the six parameters of the full DT, thus reducing sensitivity. This study seeks to validate a simplified four-parameter DT model (zeppelin) for obtaining more robust and sensitive in vivo DTI biomarkers of cartilage. METHODS: We use simulations in a substrate to mimic changes during OA; and analytic simulations of the DT drawn from a range of fractional anisotropies (FA) measured with high-quality DT data from ex vivo human cartilage. We also use in vivo data from the knees of a healthy subject and two OA patients with Kellgren-Lawrence (KL) grades 1 and 2. RESULTS: For simulated in vivo cartilage SNR ( approximately 25) and anisotropy levels, the estimated mean values of MD from the DT and zeppelin models were identical to the ground truth values. However, zeppelin's FA is more accurate in measuring water restriction. More specifically, the FA estimations of the DT model were additionally biased by between +2% and +48% with respect to zeppelin values. Additionally, both mean diffusivity (MD) and FA of the zeppelin had lower parameter variance compared to the full DT (F-test, P < 0.05). We observe the same trends from in vivo values of patient data. CONCLUSION: The zeppelin is more robust than the full DT for cartilage diffusion anisotropy and SNR at levels typically encountered in clinical applications of articular cartilage. Magn Reson Med, 2017. (c) 2017 International Society for Magnetic Resonance in Medicine.

The SNF1/AMP-activated protein kinases (AMPKs) function in energy regulation in eukaryotic cells. SNF1/AMPKs are αβγ heterotrimers that are activated by phosphorylation of the activation loop Thr on the catalytic subunit. Protein kinases that activate SNF1/AMPK have been identified, but the protein phosphatases responsible for dephosphorylation of the activation loop are less well defined. For Saccharomyces cerevisiae SNF1/AMPK, Reg1-Glc7 protein phosphatase 1 and Sit4 type 2A-related phosphatase function together to dephosphorylate Thr-210 on the Snf1 catalytic subunit during growth on high concentrations of glucose; reg1Δ and sit4Δ single mutations do not impair dephosphorylation when inappropriate glycogen synthesis, also caused by these mutations, is blocked. We here present evidence that Ptc1 protein phosphatase 2C also has a role in dephosphorylation of Snf1 Thr-210 in vivo. The sit4Δ ptc1Δ mutant exhibited partial defects in regulation of the phosphorylation state of Snf1. The reg1Δ ptc1Δ mutant was viable only when expressing mutant Snf1 proteins with reduced kinase activity, and Thr-210 phosphorylation of the mutant SNF1 heterotrimers was substantially elevated during growth on high glucose. This evidence, together with findings on the reg1Δ sit4Δ mutant, indicates that although Reg1-Glc7 plays the major role, all three phosphatases contribute to maintenance of the Snf1 activation loop in the dephosphorylated state during growth on high glucose. Ptc1 has overlapping functions with Reg1-Glc7 and Sit4 in glucose regulation of SNF1/AMPK and cell viability.

Yeast flocculation and invasive growth are processes of great interest in fundamental biology and also relevant in biotechnology and medicine. Hal3 and Vhs3 are moonlighting proteins acting in Saccharomyces cerevisiae both as inhibitors of the Ppz protein phosphatases and as components of a catalytic step in CoA biosynthesis. The double hal3 vhs3 mutant is not viable but, under semi-permissive conditions, the tetO:HAL3 vhs3 strain shows a flocculent phenotype, invasive growth and increased expression of the flocculin-encoding FLO11 gene. We show here that all these effects are caused by hyperactivation of Ppz1 as a result of depletion of its natural inhibitors. The evidence indicates that hyperactivation of Ppz1 would impair potassium transport through the Trk1/Trk2 transporters, thus resulting in a decrease in the intracellular pH and a subsequent increase in the levels of cAMP. Mutation of the TPK2 isoform of protein kinase A blocks the increase in FLO11 expression, and eliminates the flocculent and invasive phenotypes produced by depletion of Hal3 and Vhs3. Interestingly, mutation of RIM101 also significantly decreases FLO11 expression under these conditions. Cells lacking Trk1,2 display an invasive phenotype that is abolished by deletion of FLO8 or by increasing the potassium concentration in the medium. Therefore, our results support a model in which hyperactivation of Ppz phosphatases would result in alteration of potassium transport, activation of Tpk2 and signaling to the FLO11 promoter by means of the Flo8 transcription factor, thus modulating flocculation and invasive growth. This model highlights an unsuspected link between potassium homeostasis and these important morphogenetic events.

The SNF1/AMP-activated protein kinases are αβγ-heterotrimers that sense and regulate energy status in eukaryotes. They are activated by phosphorylation of the catalytic Snf1/α subunit, and the Snf4/γ regulatory subunit regulates phosphorylation through adenine nucleotide binding. In Saccharomyces cerevisiae, the Snf1 subunit is phosphorylated on the activation-loop Thr-210 in response to glucose limitation. To assess the requirement of the heterotrimer for regulated Thr-210 phosphorylation, we examined Snf1 and a truncated Snf1 kinase domain (residues 1-309) that has partial Snf1 function. Snf1(1-309) does not interact with the β and Snf4/γ regulatory subunits, and its activity was independent of them in vivo. Phosphorylation of both Snf1 and Snf1(1-309) increased in response to glucose limitation in wild-type cells and in cells lacking β- and Snf4/γ-subunits. These results indicate that glucose regulation of activation-loop phosphorylation can occur by mechanism(s) that function independently of the regulatory subunits. We further show that the Reg1-Glc7 protein phosphatase 1 and Sit4 type 2A-like phosphatase are largely responsible for dephosphorylation of Thr-210 of Snf1(1-309). Together, these findings suggest that these two phosphatases mediate heterotrimer-independent regulation of Thr-210 phosphorylation.

Alkaline pH stress invokes a potent and fast transcriptional response in Saccharomyces cerevisiae that includes many genes repressed by glucose. Certain mutants in the glucose-sensing and -response pathways, such as those lacking the Snf1 kinase, are sensitive to alkalinization. In the present study we show that the addition of glucose to the medium improves the growth of wild-type cells at high pH, fully abolishes the snf1 alkali-sensitive phenotype and attenuates high pH-induced Snf1 phosphorylation at Thr(210). Lack of Elm1, one of the three upstream Snf1 kinases (Tos3, Elm1 and Sak1), markedly increases alkali sensitivity, whereas the phenotype of the triple mutant tos3 elm1 sak1 is even more pronounced than that of snf1 cells and is poorly rescued by glucose supplementation. DNA microarray analysis reveals that about 75% of the genes induced in the short term by high pH are also induced by glucose scarcity. Snf1 mediates, in full or in part, the activation of a significant subset (38%) of short-term alkali-induced genes, including those encoding high-affinity hexose transporters and phosphorylating enzymes. The induction of genes encoding enzymes involved in glycogen, but not trehalose, metabolism is largely dependent of the presence of Snf1. Therefore the function of Snf1 in adaptation to glucose scarcity appears crucial for alkaline pH tolerance. Incorporation of micromolar amounts of iron and copper to a glucose-supplemented medium resulted in an additive effect and allows near-normal growth at high pH, thus indicating that these three nutrients are key limiting factors for growth in an alkaline environment.

Ypi1 was discovered as an essential protein able to act as a regulatory subunit of the Saccharomyces cerevisiae type 1 protein phosphatase Glc7 and play a key role in mitosis. We show here that partial depletion of Ypi1 causes lithium sensitivity and that high levels of this protein confer a lithium-tolerant phenotype to yeast cells. Remarkably, this phenotype was independent of the role of Ypi1 as a Glc7 regulatory subunit. Lithium tolerance in cells overexpressing Ypi1 was caused by a combination of increased efflux of lithium, mediated by augmented expression of the alkaline cation ATPase ENA1, and decreased lithium influx through the Trk1,2 high-affinity potassium transporters. Deletion of CNB1, encoding the regulatory subunit of the calcineurin phosphatase, blocked Ypi1-induced expression of ENA1, normalized Li(+) fluxes, and abolished the Li(+) hypertolerant phenotype of Ypi1-overexpressing cells. These results point to a complex role of Ypi1 on the regulation of cation homeostasis, largely mediated by the calcineurin phosphatase.