Reliability of Semi-Automated Segmentations in Glioblastoma.

PURPOSE: In glioblastoma, quantitative volumetric measurements of contrast-enhancing or fluid-attenuated inversion recovery (FLAIR) hyperintense tumor compartments are needed for an objective assessment of therapy response. The aim of this study was to evaluate the reliability of a semi-automated, region-growing segmentation tool for determining tumor volume in patients with glioblastoma among different users of the software. METHODS: A total of 320 segmentations of tumor-associated FLAIR changes and contrast-enhancing tumor tissue were performed by different raters (neuroradiologists, medical students, and volunteers). All patients underwent high-resolution magnetic resonance imaging including a 3D-FLAIR and a 3D-MPRage sequence. Segmentations were done using a semi-automated, region-growing segmentation tool. Intra- and inter-rater-reliability were addressed by intra-class-correlation (ICC). Root-mean-square error (RMSE) was used to determine the precision error. Dice score was calculated to measure the overlap between segmentations. RESULTS: Semi-automated segmentation showed a high ICC (> 0.985) for all groups indicating an excellent intra- and inter-rater-reliability. Significant smaller precision errors and higher Dice scores were observed for FLAIR segmentations compared with segmentations of contrast-enhancement. Single rater segmentations showed the lowest RMSE for FLAIR of 3.3 % (MPRage: 8.2 %). Both, single raters and neuroradiologists had the lowest precision error for longitudinal evaluation of FLAIR changes. CONCLUSIONS: Semi-automated volumetry of glioblastoma was reliably performed by all groups of raters, even without neuroradiologic expertise. Interestingly, segmentations of tumor-associated FLAIR changes were more reliable than segmentations of contrast enhancement. In longitudinal evaluations, an experienced rater can detect progressive FLAIR changes of less than 15 % reliably in a quantitative way which could help to detect progressive disease earlier.

Bilateral cartilage T2 mapping 9 years after Mega-OATS implantation at the knee: a quantitative 3T MRI study.

OBJECTIVE: To evaluate morphological and quantitative MR findings 9 years after autograft transfer of the posterior femoral condyle (Mega-OATS) and to correlate these findings with clinical outcomes. Quantitative MR measurements were also obtained of the contralateral knee and the utility as reference standard was investigated. DESIGN: Both knees of 20 patients with Mega-OATS osteochondral repair at the medial femoral condyle (MFC) were studied using 3T MRI 9 years after the procedure. MR-sequences included morphological sequences and a 2D multislice multiecho (MSME) spin echo (SE) sequence for quantitative cartilage T2 mapping. Cartilage segmentation was performed at the cartilage repair site and six additional knee compartments. Semi-quantitative MR observation of cartilage repair tissue (MOCART) scores and clinical Lysholm scores were obtained. Paired t-tests and Spearman correlations were used for statistical analysis. RESULTS: Global T2-values were significantly higher at ipsilateral knees compared to contralateral knees (42.1 ± 3.0 ms vs 40.4 ± 2.6 ms, P = 0.018). T2-values of the Mega-OATS site correlated significantly with MOCART scores (R = -0.64, P = 0.006). The correlations between MOCART and Lysholm scores and between absolute T2-values and Lysholm scores were not significant (P > 0.05). However, higher T2 side-to-side differences at the femoral condyles correlated significantly with more severe clinical symptoms (medial, R = -0.53, P = 0.030; lateral, R = -0.51, P = 0.038). CONCLUSIONS: Despite long-term survival, 9 years after Mega-OATS procedures, T2-values of the grafts were increased compared to contralateral knees. Clinical scores correlated best with T2 side-to-side differences of the femoral condyles, indicating that intraindividual adjustment may be beneficial for outcome evaluation.

Double inversion recovery sequence of the cervical spinal cord in multiple sclerosis and related inflammatory diseases.

BACKGROUND AND PURPOSE: MR imaging plays an important role in diagnosing MS and other related inflammatory diseases; however, imaging of the spinal cord is still challenging. We hypothesized that a 3D double inversion recovery sequence for cervical spinal cord imaging would be more sensitive in detecting inflammatory lesions than a conventional 2D T2-weighted TSE sequence at 3T. MATERIALS AND METHODS: On a 3T MR imaging scanner, we examined 30 patients with suspected or established MS (MS, n = 16; clinically isolated syndrome, n = 12; isolated myelitis, n = 2) and 10 healthy controls. Newly developed 3D double inversion recovery and conventional 2D axial and sagittal T2-weighted TSE images of the cervical spinal cord were acquired. Two blinded neuroradiologists independently assessed the scans in pseudorandomized order for lesion numbers and rated lesion visibility and overall image quality on 5-point scales. A subsequent consensus reading delivered definite lesion counts. Standardized contrast-to-noise ratios were calculated in representative lesions of each patient. RESULTS: Overall, 28% more lesions could be detected with 3D double inversion recovery than with conventional T2WI (119 versus 93, P < .002). On average, the standardized contrast-to-noise ratio was significantly higher (P < .001) in double inversion recovery than in T2WI. Lesion visibility was rated significantly higher (P < .001) in double inversion recovery compared with T2WI despite lower image quality. CONCLUSIONS: The novel 3D double inversion recovery sequence allowed better detection of lesions in MS and related inflammatory diseases of the cervical spinal cord, compared with conventional 2D T2WI.

Bone mineral density values derived from routine lumbar spine multidetector row CT predict osteoporotic vertebral fractures and screw loosening.

BACKGROUND AND PURPOSE: Established methods of assessing bone mineral density are associated with additional radiation exposure to the patient. In this study, we aimed to validate a method of assessing bone mineral density in routine multidetector row CT of the lumbar spine. MATERIALS AND METHODS: In 38 patients, bone mineral density was assessed in quantitative CT as a standard of reference and in sagittal reformations derived from standard multidetector row CT studies without IV contrast. MDCT-to-quantitative CT conversion equations were calculated and then applied to baseline multidetector row scans of another 62 patients. After a mean follow-up of 15 ± 6 months, patients were re-assessed for incidental fractures and screw loosening after spondylodesis (n = 49). RESULTS: We observed conversion equations bone mineral densityMDCT = 0.78 × Hounsfield unitMDCTmg/mL (correlation with bone mineral densityquantitative CT, R(2) = 0.92, P < .001) for 120 kV(peak) tube voltage and bone mineral densityMDCT = 0.86 × Hounsfield unitMDCTmg/mL (R(2) = 0.81, P < .001) for 140 kVp, respectively. Seven patients (11.3%) had existing osteoporotic vertebral fractures at baseline, while 8 patients (12.9%) showed incidental osteoporotic vertebral fractures. Screw loosening was detected in 28 patients (57.1% of patients with spondylodesis). Patients with existing vertebral fractures showed significantly lower bone mineral densityMDCT than patients without fractures (P < .01). At follow-up, patients with incidental fractures and screw loosening after spondylodesis, respectively, showed significantly lower baseline bone mineral densityMDCT (P < .001 each). CONCLUSIONS: This longitudinal study demonstrated that converted bone mineral density values derived from routine lumbar spine multidetector row CT adequately differentiated patients with and without osteoporotic fractures and could predict incidental fractures and screw loosening after spondylodesis.

Alignment does not influence cartilage T2 in asymptomatic knee joints.

PURPOSE: To investigate whether the static knee alignment affects articular cartilage ultrastructures when measured using T2 relaxation among asymptomatic subjects. METHODS: Both knee joints (n = 96) of 48 asymptomatic volunteers (26 females, 22 males; 25.4 ± 1.7 years; no history of major knee trauma or surgery) were evaluated clinically (Lysholm, Tegner) and by MRI (hip-knee-ankle angle, standard knee protocol, T2 mapping). Group (n = 4) division was as follows: neutral (<1° varus/valgus), mild varus (2°-4° varus), severe varus (>4° varus) and valgus (2°-4° valgus) deformity with n = 12 subjects/group; n = 24 knees/group. Regions of interest (ROI) for T2 assessment were placed within full-thickness cartilage across the whole joint surface and were divided respecting compartmental as well as functional joint anatomy. RESULTS: Leg alignment was 0.7° ± 0.5° varus among neutral, 3.0° ± 0.6° varus among mild varus, 5.0° ± 1.1° varus among severe varus and 2.5° ± 0.7° valgus among valgus group subjects and thus significantly different. No differences between the groups emerged from clinical measures. No morphological pathology was detected in any knee joint. Global T2 values (42.3 ± 2.3; 37.7-47.9 ms) of ROIs placed within every knee joint per subject were not different between alignment groups or between genders, respectively. CONCLUSION: Static frontal plane leg malalignment does not affect cartilage ultrastructure among young, asymptomatic individuals as measured by T2 quantitative imaging. LEVEL OF EVIDENCE: Cross-sectional study, Level II-III.

High-resolution bone imaging for osteoporosis diagnostics and therapy monitoring using clinical MDCT and MRI.

Osteoporosis is classified as a public health problem due to its increased risk for fragility fractures. Osteoporotic fractures, in particular spine and hip fractures, are associated with a high morbidity and mortality, and generate immense financial cost. The World Health Organisation (WHO) based the diagnosis of osteoporosis on the measurement of bone mineral density (BMD) using dual-energy X-ray absorptiometry (DXA). However, BMD values of subjects with versus without osteoporotic fractures overlap. Furthermore, it was reported that the anti-fracture effects of drugs could be only partially explained by their effects on BMD. Bone strength reflects the integration of BMD and bone quality. The later can be partly determined by measurements of bone microstructure. Therefore, substantial research efforts have been undertaken to assess bone microstructure by using high-resolution imaging techniques, including high-resolution peripheral quantitative computed tomography (hr-pQCT), high-resolution multi-detector computed tomography (MDCT), and high-resolution magnetic resonance imaging (MRI). Clinical MDCT and MRI systems are broadly available and allow an adequate depiction of the bone microstructure at the clinically most important fracture sites, i.e. radius, spine and hip. Bone microstructure parameters and finite element models can be computed in high-resolution MDCT and MR images. These measurements improved the prediction of bone strength beyond the DXA-derived BMD and revealed pharmacotherapy effects, which are partly not captured by BMD. Therefore, high-resolution bone imaging using clinical MDCT and MRI may be beneficial for osteoporosis diagnostics and allow a highly sensitive monitoring of drug treatment, which plays an important role in the prevention of fragility fractures.

Cartilage and meniscal T2 relaxation time as non-invasive biomarker for knee osteoarthritis and cartilage repair procedures.

OBJECTIVE: The purpose of this work was to review the current literature on cartilage and meniscal T2 relaxation time. METHODS: Electronic searches in PubMed were performed to identify relevant studies about T2 relaxation time measurements as non-invasive biomarker for knee osteoarthritis (OA) and cartilage repair procedures. RESULTS: Initial osteoarthritic changes include proteoglycan loss, deterioration of the collagen network, and increased water content within the articular cartilage and menisci. T2 relaxation time measurements are affected by these pathophysiological processes. It was demonstrated that cartilage and meniscal T2 relaxation time values were significantly increased in subjects with compared to those without radiographic OA and focal knee lesions, respectively. Subjects with OA risk factors such as overweight/obesity showed significantly greater cartilage T2 values than normal controls. Elevated cartilage and meniscal T2 relaxation times were found in subjects with vs without knee pain. Increased cartilage T2 at baseline predicted morphologic degeneration in the cartilage, meniscus, and bone marrow over 3 years. Furthermore, cartilage repair tissue could be non-invasively assessed by using T2 mapping. Reproducibility errors for T2 measurements were reported to be smaller than the T2 differences in healthy and diseased cartilage indicating that T2 relaxation time may be a reliable discriminatory biomarker. CONCLUSIONS: Cartilage and meniscal T2 mapping may be suitable as non-invasive biomarker to diagnose early stages of knee OA and to monitor therapy of OA.

Bone mineral density measurements of the proximal femur from routine contrast-enhanced MDCT data sets correlate with dual-energy X-ray absorptiometry.

OBJECTIVES: To evaluate the utility of femoral bone mineral density (BMD) measurements in routine contrast-enhanced multi-detector computed tomography (ceMDCT) using dual-energy X-ray absorptiometry (DXA) as the reference standard. METHODS: Forty-one patients (33 women, 8 men) underwent DXA measurement of the proximal femur. Subsequently, transverse sections of routine ceMDCT of these patients were used to measure BMD of the femoral head and femoral neck. The MDCT-to-DXA conversion equations for BMD and T-score were calculated using linear regression analysis. The conversion equations were applied to the MDCT data sets of 382 patients (120 women, 262 men) of whom 74 had osteoporotic fractures. RESULTS: A correlation coefficient of r = 0.84 (P < 0.05) was calculated for BMD(MDCT) values of the femoral head and DXA T-scores of the total proximal femur using the conversion equation T-score = 0.021 × BMD(MDCT) - 5.90. The correlation coefficient for the femoral neck was r = 0.79 (P < 0.05) with the conversion equation T-score = 0.016 × BMD(MDCT) - 4.28. Accordingly, converted T-scores for the femoral neck in patients with versus those without osteoporotic fractures were significantly different (female, -1.83 versus -1.47; male, -1.86 versus -1.47; P < 0.05). CONCLUSION: BMD measurements of the proximal femur were computed in routine contrast-enhanced MDCT and converted to DXA T-scores, which adequately differentiated patients with and without osteoporotic fractures.

Clinical pilot study for the automatic segmentation and recognition of abdominal adipose tissue compartments from MRI data.

PURPOSE: In the diagnosis and risk assessment of obesity, both the amount and distribution of adipose tissue compartments are critical factors. We present a hybrid method for the quantitative measurement of human body fat compartments. MATERIALS AND METHODS: MRI imaging was performed on a 1.5 T scanner. In a pre-processing step, the images were corrected for bias field inhomogeneity. For segmentation and recognition a hybrid algorithm was developed to automatically differentiate between different adipose tissue compartments. The presented algorithm is designed with a combination of shape and intensity-based techniques. To incorporate the presented algorithm into the clinical routine, we developed a graphical user interface. Results from our methods were compared with the known volume of an adipose tissue phantom. To evaluate our method, we analyzed 40 clinical MRI scans of the abdominal region. RESULTS: Relatively low segmentation errors were found for subcutaneous adipose tissue (3.56 %) and visceral adipose tissue (0.29 %) in phantom studies. The clinical results indicated high correlations between the distribution of adipose tissue compartments and obesity. CONCLUSION: We present an approach that rapidly identifies and quantifies adipose tissue depots of interest. With this method examination and analysis can be performed in a clinically feasible timeframe.

Automated 3D trabecular bone structure analysis of the proximal femur--prediction of biomechanical strength by CT and DXA.

SUMMARY: The standard diagnostic technique for assessing osteoporosis is dual X-ray absorptiometry (DXA) measuring bone mass parameters. In this study, a combination of DXA and trabecular structure parameters (acquired by computed tomography [CT]) most accurately predicted the biomechanical strength of the proximal femur and allowed for a better prediction than DXA alone. INTRODUCTION: An automated 3D segmentation algorithm was applied to determine specific structure parameters of the trabecular bone in CT images of the proximal femur. This was done to evaluate the ability of these parameters for predicting biomechanical femoral bone strength in comparison with bone mineral content (BMC) and bone mineral density (BMD) acquired by DXA as standard diagnostic technique. METHODS: One hundred eighty-seven proximal femur specimens were harvested from formalin-fixed human cadavers. BMC and BMD were determined by DXA. Structure parameters of the trabecular bone (i.e., morphometry, fuzzy logic, Minkowski functionals, and the scaling index method [SIM]) were computed from CT images. Absolute femoral bone strength was assessed with a biomechanical side-impact test measuring failure load (FL). Adjusted FL parameters for appraisal of relative bone strength were calculated by dividing FL by influencing variables such as body height, weight, or femoral head diameter. RESULTS: The best single parameter predicting FL and adjusted FL parameters was apparent trabecular separation (morphometry) or DXA-derived BMC or BMD with correlations up to r = 0.802. In combination with DXA, structure parameters (most notably the SIM and morphometry) added in linear regression models significant information in predicting FL and all adjusted FL parameters (up to R(adj) = 0.872) and allowed for a significant better prediction than DXA alone. CONCLUSION: A combination of bone mass (DXA) and structure parameters of the trabecular bone (linear and nonlinear, global and local) most accurately predicted absolute and relative femoral bone strength.

T2 assessment and clinical outcome following autologous matrix-assisted chondrocyte and osteochondral autograft transplantation.

OBJECTIVE: Both, matrix-assisted chondrocyte transplantation (MACT) and osteochondral autograft transplantation (OCT), are applied for treatment of articular cartilage defects. While previous clinical studies have compared the respective outcome, there is no such information investigating the ultrastructural composition using T2 mapping comparing cartilage T2 values of the repair tissue (RT). METHODS: Eighteen patients that underwent MACT or OCT for treatment of cartilage defects at the knee joint (nine MACT, nine OCT) were matched for gender (one female, eight male pairs), age (33.8), body mass index (BMI) (28.3), defect localization, and postoperative interval (41.6 months). T2 assessment was accomplished by T2 maps, while the clinical evaluation included the Lysholm and Cincinnati knee scores, a visual analogue scale (VAS) for pain, the Tegner activity scale, and the Short Form-36. RESULTS: Global T2 values of healthy femoral cartilage (HC) were similar among groups, while T2 values of the RT following MACT (46.8ms, SD 8.6) were significantly lower when compared to RT T2 values after OCT (55.5ms, SD 6.7) (P=0.048). MACT values were also significantly lower in comparison to HC (52.5ms, SD 7.9) within MACT patients (P=0.046), while OCT values were significantly higher compared to HC (49.9ms, SD 5.1) within OCT patients (P=0.041). The clinical outcome following MACT was consistently superior to that after OCT while only the Lysholm score reached the level of significance (MACT 77.0, OCT 66.8; P=0.04). CONCLUSION: These findings indicate that MACT and OCT result in a different ultrastructural outcome, which is only partially represented by the clinical picture.

[Fracture diagnosis in osteoporosis]. / Frakturdiagnostik in der Osteoporose.

As life expectancy rises the prevalence of osteoporosis also increases, which represents a growing burden for the populace and the healthcare system. Vertebral fractures are the most frequent type of all osteoporotic fractures. Since they can be diagnosed by radiology, these examinations are particularly important for devising suitable treatment strategies. The goal of activities undertaken by osteoporosis organizations is to put across to radiologists the importance of accurate evaluation of these fractures. Identification of osteoporotic vertebral fractures is however problematic since the transition from healthy vertebral bodies to those deformed by osteoporosis constitutes a continuum and it is often difficult in deformed vertebral bodies to distinguish between an osteoporotic fracture and a non-osteoporotic deformity. This overview attempts to heighten awareness of the significant role the radiologist plays in the diagnostic work-up of osteoporosis and to provide diagnostic aids for assessing osteoporotic vertebral fractures including differential diagnoses.

Structural analysis of trabecular bone of the proximal femur using multislice computed tomography: a comparison with dual X-ray absorptiometry for predicting biomechanical strength in vitro.

We investigated whether trabecular microstructural parameters determined in multislice spiral computed tomographic (MSCT) images of proximal femur specimens differed in male and female donors and improved the prediction of biomechanical strength of the femur compared to bone mineral density (BMD) and content (BMC) determined with dual X-ray absorptiometry (DXA) as the standard diagnostic technique. Proximal femur specimens (n = 119) were harvested from formalin-fixed human cadavers (mean age 80 +/- 10 years). BMD was determined using DXA. Trabecular microstructural parameters (bone volume fraction, fractal dimension, and trabecular thickness, spacing, and number) were calculated in MSCT-derived images of the proximal femur. Failure load (FL) was measured using a biomechanical side-impact test. An age-, height-, and weight-matched subgroup (n = 54) was chosen to compare male and female donors. BMC, BMD, and structural parameters correlated significantly with FL, with r up to 0.75, 0.71, and 0.71, respectively. In a multiple regression model, an increase up to r = 0.82 was obtained when combining trabecular structural parameters and BMC. BMD differed between males and females only at the trochanter. BMC showed significant gender differences in all regions. This experimental study showed that a combination of BMC and microstructural parameters could improve the prediction of FL, suggesting that bone mass and trabecular structure carry overlapping but complementary information and that a combination of the two provides the best prediction of bone strength. Male donors had larger femora even after adjustment for body size and height, but no differences in trabecular structure were found between males and females.

Detection of osteoporotic vertebral fractures using multidetector CT.

INTRODUCTION: Goals were to compare the performance of lateral radiographs and sagittal reformations (SR) of axial computed tomography (CT) datasets in identification of osteoporotic vertebral fractures and to assess for optimal slice thickness in axial CT datasets needed for reliable classification of these fractures. METHODS: Sixty-five vertebrae were harvested from 21 human cadaver spines and examined with a 64-row multidetector CT scanner. Axial images were acquired with a slice thickness of 0.6, 1, 2, 3 and 5 mm and SR were obtained using these datasets. In addition, specimens were radiographed in antero-posterior and lateral orientation. Vertebrae visualized in the different image datasets were separately graded by four radiologists according to the spinal fracture index (SFI) classification. Fracture status determined in a consensus reading of interactive reformations of the 0.6-mm CT dataset in all three dimensions served as a standard of reference in combination with pathological examinations. RESULTS: The average agreement for the 0.6-mm SR obtained between each radiologist and standard of reference for the grading of the fractures was very good (kappa=0.81). It was good for the 1-, 2- and 3-mm SR (kappa=0.70, 0.69 and 0.64), but only moderate for the radiographs (kappa=0.52), and fair for the 5-mm SR (kappa=0.33). When focusing only on detection of fractures, independent of the grading, all kappa values improved by about 0.15, resulting in excellent values for the 0.6-mm through 3-mm SR (0.95

[Multislice-CT for structure analysis of trabecular bone - a comparison with micro-CT and biomechanical strength]. / Mehrschicht-CT zur Strukturanalyse des trabekulären Knochens - Vergleich mit Mikro-CT und biomechanischer Festigkeit.

OBJECTIVES: MS-CT (Multislice-Spiral-CT) has a higher spatial resolution compared to the SS-CT (Singleslice-CT). The purpose of this study was to investigate, if the higher spatial resolution of the MS-CT has advantages for structural analyses in the assessment of osteoporosis. MATERIAL AND METHODS: 20 cylindrical trabecular bone specimens (diameter 12 mm, length 15 - 20 mm) were harvested from formalin-fixed human thoracic spines. All specimens were examined by Micro-CT and quantitative, histomorphologic parameters were determined. Analogous structural parameters were calculated from the high-resolution images acquired by both MS- and SS-CT. Additionally, the BMD (bone mineral density) was measured by QCT (quantitative CT). The maximum compressive strength (MCS) was determined in a biomechanical test. The structural parameters were correlated with the histomorphologic parameters and with the MCS. RESULTS: The parameters bone fraction and trabecular separation correlated significantly in both MS- and SS-CT with the analogous parameters from Micro-CT (r (2) = 0.84, p < 0.01) and the MCS (r (2) = 0.81, p < 0.01). The highest correlation with the MCS was calculated using the trabecular number measured by MS-CT in the superior region near the endplate of the vertebra with the high-resolution kernel U90 u (r (2) = 0.85, p < 0.01). This correlation was significantly higher than the correlation between MCS and BMD (r (2) = 0.49, p < 0.01). CONCLUSION: Micro-CT- and MS-CT-determined structural parameters of the trabecular bone showed significant, high correlations. Thus, a characterisation of the trabecular structure seems to be possible. The biomechanical stability of the bone can also be predicted well. The structural parameters acquired by MS-CT show higher correlations with the MCS than the BMD or structural parameters determined by SS-CT do. In this study MS-CT was best suited to predict biomechanical strength of trabecular bone.

The alpha 5 beta 1 integrin fibronectin receptor, but not the alpha 5 cytoplasmic domain, functions in an early and essential step in fibronectin matrix assembly.

The alpha 5 beta 1 integrin mediates cell adhesion and migration on fibronectin, a glycoprotein critical for normal vertebrate embryonic development. Indirect evidence reported to date suggests that this receptor also functions in the deposition of fibronectin matrices. We used a molecular genetic approach to critically evaluate this role of alpha 5 beta 1 integrins. Mutant Chinese hamster ovary (CHO) cells deficient in alpha 5 integrin expression could not assemble a fibronectin matrix. Reconstituting alpha 5 beta 1 integrin expression by transfecting them with a full-length cDNA encoding the human alpha 5 chain completely restored fibronectin matrix assembly. CHO cells expressing an alpha 5 chain lacking the cytoplasmic domain also assembled a fibronectin matrix. Removing the cytoplasmic domain of alpha 5 appears to increase its activity in fibronectin matrix assembly. In addition to alpha 5 beta 1 integrin binding to fibronectin's RGD-containing domain, cells must bind with high affinity to fibronectin's amino-terminal 29-kDa matrix assembly domain to form a fibronectin matrix. Studies with the alpha 5-deficient CHO cells show that the expression of alpha 5 beta 1 integrin is also necessary for cells to bind fragments containing this distinct site in fibronectin and that a fibronectin matrix increases binding of the 29-kDa fragment. Thus, alpha 5 beta 1 integrins not only mediate cell adhesion to fibronectin, but also play an essential role in the assembly of a fibronectin matrix. This role includes direct binding to fibronectin and modulating a distinct binding event involving the interaction of fibronectin's amino-terminal matrix assembly domain with the cell surface.

Functional role of the cytoplasmic domain of the integrin alpha 5 subunit.

The purpose of this study was to explore the functional role of the cytoplasmic domain of the alpha subunit of the alpha 5/beta 1 integrin, a fibronectin receptor. Mutant CHO cells that express very low levels of endogenous hamster alpha 5 subunit (CHO clone B2) were transfected with an expression vector containing full-length or truncated human alpha 5 cDNAs to form chimeric human alpha 5/hamster beta 1 integrins. Three transfectants were examined: B2a27 expresses a full-length human alpha 5 subunit with 27 amino acids in the cytoplasmic domain; B2a10 expresses an alpha 5 with a 17-amino acid cytoplasmic truncation; B2a1 expresses an alpha 5 with a 26-amino acid truncation. Levels of alpha 5/beta 1 surface expression in B2a27 and B2a10 cells were similar to that in wild type CHO cells. The expression of alpha 5/beta 1 in B2a1 cells was less, amounting to 15-20% of WT levels, despite message levels that were three to five times greater than those of B2a27. The transfectants were used to examine the role of the alpha 5 cytoplasmic domain in cell adhesion, cell motility, cytoskeletal organization, and integrin-mediated tyrosine phosphorylation. The adhesion characteristics of B2a27 and B2a10 cells on fibronectin substrata were similar to each other and to wild type CHO cells. B2a1 cells displayed slight reductions in the strength and rate of adhesion to fibronectin. Cell motility in the presence of fibronectin was similar for B2a27, B2a10, and wild type CHO cells, while the B2a1 cells were substantially less motile. Comparable degrees of cell spreading and extensive organization of actin filaments were observed for B2a27, B2a10, and wild type CHO cells on fibronectin substrata. The B2a1 cells spread to a lesser degree, and some organization of actin was observed; the untransfected B2 cells remained round on fibronectin substrata and showed no actin reorganization. Since the reduced motility and cell spreading observed in the B2a1 cells might be due either to reduced surface expression of alpha 5/beta 1 or to the truncation in the alpha 5 cytoplasmic domain, we used flow cytometric cell sorting to select populations of B2a1 and B2a27 cells expressing similar levels of cell surface alpha 5. The deficits in spreading and motility were present in B2a1 cells expressing high levels of alpha 5. Thus the region of the alpha 5 cytoplasmic domain adjacent to the membrane seems to play an important role in cytoskeletal organization and cell motility. We also examined whether alpha subunit truncation would affect integrin-mediated tyrosine phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)

The alpha v beta 1 integrin functions as a fibronectin receptor but does not support fibronectin matrix assembly and cell migration on fibronectin.

The fibronectin receptor, alpha 5 beta 1, has been shown to be required for fibronectin matrix assembly and plays an important role in cell migration on fibronectin. However, it is not clear whether other fibronectin binding integrins can take the place of alpha 5 beta 1 during matrix assembly and cell migration. To test this, we expressed the human alpha v subunit in the CHO cell line CHO-B2 that lacks the alpha 5 subunit. We found that the human alpha v combined with CHO cell beta 1 to form the integrin alpha v beta 1. Cells that expressed alpha v beta 1 attached to and spread well on fibronectin-coated dishes, but did so less well on vitronectin-coated dishes. This, along with other data, indicated that alpha v beta 1 functions as a fibronectin receptor in CHO-B2 cells. The alpha v beta 1-expressing cells failed to produce a fibronectin matrix or to migrate on fibronectin, although the same cells transfected with alpha 5 do produce a matrix and migrate on fibronectin. The affinity of the alpha v beta 1-expressing cells for fibronectin was fourfold lower than that of the alpha 5 beta 1-expressing cells. In addition, alpha v beta 1 was distributed diffusely throughout the cell surface, whereas alpha 5 beta 1 was localized to focal adhesions when cells were seeded onto fibronectin-coated surfaces. Thus, of the two fibronectin receptors, alpha v beta 1 and alpha 5 beta 1, only alpha 5 beta 1 supports fibronectin matrix assembly and promotes cell migration on fibronectin in the CHO-B2 cells. Possible reasons for this difference in the activities of alpha v beta 1 and alpha 5 beta 1 include the lower affinity of alpha v beta 1 for fibronectin and the failure of this integrin to localize in adhesion plaques on a fibronectin substrate. These results show that two integrins with similar ligand specificities and cell attachment functions may be quite different in their ability to support fibronectin matrix assembly and cell motility on fibronectin.

Motility of fibronectin receptor-deficient cells on fibronectin and vitronectin: collaborative interactions among integrins.

Cells are capable of adhering to and migrating on protein components of the extracellular matrix. These cell-matrix interactions are thought to be mediated largely through a family of cell surface receptors termed integrins. However, the manner in which individual integrins are involved in cell adhesion and motility has not been fully determined. To explore this issue, we previously selected a series of CHO variants that are deficient in expression of the integrin alpha 5 beta 1, the "classical" fibronectin receptor. Two sets of subclones of these variants were defined which respectively express approximately 20% or 2% of fibronectin receptor on the cell surface when compared to wild-type cells (Schreiner, C. L., J. S. Bauer, Y. N. Danilov, S. Hussein, M. M. Sczekan, and R. L. Juliano. 1989. J. Cell Biol. 109:3157-3167). In the current study, the variant clones were tested for haptotactic motility on substrata coated with fibronectin or vitronectin. Data from assays using fibronectin show that cellular motility of the 20% variants was substantially decreased (30-75% of wild type), while the motility of the 2% variants was nearly abolished (2-20% of wild type). Surprisingly, a similar pattern was seen for haptotactic motility of both 2% and 20% variants when vitronectin was used (approximately 20-30% of wild type). The reduced haptotactic motility of the fibronectin receptor-deficient variant clones on vitronectin was shown not to be due to reduced vitronectin receptor (alpha v beta 3) expression nor to a failure of these variants to adhere to vitronectin substrata. Transfection of the deficient variants with a cDNA for the human alpha 5 subunit resulted in normal levels of fibronectin receptor expression (as a human alpha 5/hamster beta 1 chimera) and restored the motility of the CHO variants on fibronectin and vitronectin. This indicates that expression of the alpha 5 subunit is required for normal haptotactic motility on vitronectin substrata and suggests that the fibronectin receptor (alpha 5 beta 1) plays a cooperative role with vitronectin receptors in cell motility.

Isolation and characterization of Chinese hamster ovary cell variants deficient in the expression of fibronectin receptor.

Chinese hamster ovary cell populations were enriched for cells displaying low surface expression of the 140-kD integrin fibronectin receptor (FnR) by means of fluorescence-activated cell sorting using monoclonal anti-FnR antibodies. Selected cells were cloned by limiting dilution, and the resulting clones were screened for low cell surface FnR expression by ELISA. Two multiply sorted populations gave rise to variant clones possessing approximately 20 or 2% FnR expression, respectively, compared with wild-type cells. Growth rates of the "20%" and "2%" clones on serum-coated plastic dishes were similar to that of wild-type cells. Variant cells expressing 20% FnR could attach and spread on substrata coated with purified fibronectin, although somewhat more slowly than wild-type cells, while cells expressing 2% FnR could not attach or spread. Cells from all variant clones attached normally to vitronectin substrata, but some of the 2% clones displayed altered morphology on this type of substratum. Motility assays in blind well chambers showed a correlation of movement with level of expression of FnR. The number of cells migrating in response to fibronectin was greatly reduced compared with wild-type cells for the 20% FnR variant clones, while variant clones with 2% FnR showed virtually no migratory activity. Surface labeling with 125I and immunoaffinity purification of FnR showed reduced levels of intact FnR on the plasma membranes of variants with 20% FnR, while none was detected in variants expressing 2% FnR. Nevertheless, beta subunits were detected on the surfaces of all variant clones. Immunoblots of cell lysates from wild-type cells and from both types of variant clones showed substantial amounts of FnR beta chain as well as enhanced amounts of a pre-beta moiety in the variants. alpha chain was markedly reduced in the 20% variants and essentially absent in the 2% variants, indicating that failure to assemble intact FnR in these variants was due to deficiencies of alpha chain production. Dot blots of total mRNA from a representative clone expressing 20% FnR showed reduced levels of material hybridizing to an 0.97-kb hamster FnR alpha chain cDNA probe as compared with wild type, while mRNA from a representative clone expressing 2% FnR had no detectable hybridizable RNA; this seems to agree well with the results obtained by immunoblotting. Thus, the defect in the variant clones seems to be due to reduced levels of alpha chain mRNA leading to a deficit of mature FnR and consequent alterations in cell adhesion and motility on fibronectin substrata.