Tissue vascularization through 3D printing: Will technology bring us flow?

BACKGROUND: Though in vivo models provide the most physiologically relevant environment for studying tissue function, in vitro studies provide researchers with explicit control over experimental conditions and the potential to develop high throughput testing methods. In recent years, advancements in developmental biology research and imaging techniques have significantly improved our understanding of the processes involved in vascular development. However, the task of recreating the complex, multi-scale vasculature seen in in vivo systems remains elusive. RESULTS: 3D bioprinting offers a potential method to generate controlled vascular networks with hierarchical structure approaching that of in vivo networks. Bioprinting is an interdisciplinary field that relies on advances in 3D printing technology along with advances in imaging and computational modeling, which allow researchers to monitor cellular function and to better understand cellular environment within the printed tissue. CONCLUSIONS: As bioprinting technologies improve with regards to resolution, printing speed, available materials, and automation, 3D printing could be used to generate highly controlled vascularized tissues in a high throughput manner for use in regenerative medicine and the development of in vitro tissue models for research in developmental biology and vascular diseases.

Expression of vitamin D-metabolizing enzymes in human adipose tissue -- the effect of obesity and diet-induced weight loss.

OBJECTIVE: Low vitamin D (VD) levels are common in obesity. We hypothesized that this may be due to metabolism of VD in adipose tissue (AT). Thus, we studied (1) whether the VD-metabolizing enzymes were expressed differently in AT of lean and obese individuals and in visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT), and (2) whether their expression was influenced by weight loss. METHODS: Samples of SAT and VAT were analyzed for expression of the vitamin-D-25-hydroxylases CYP2R1, CYP2J2, CYP27A1 and CYP3A4, the 25-vitamin-D-1α-hydroxylase CYP27B1, the catabolic vitamin-D-24-hydroxylase CYP24A1, and the vitamin D receptor, using reverse transcriptase-PCR. Moreover, plasma 25-hydroxy-vitamin D (25OHD) level was measured and related to the expression of these enzymes. Samples of SAT and VAT from 20 lean women and 20 obese women, and samples of SAT from 17 obese subjects before and after a 10% weight loss were analyzed. RESULTS: A plasma 25OHD level <50 nmol l(-1) was highly prevalent in both lean (45%) and obese (90%) women (P<0.01). Plasma 25OHD increased by 27% after weight loss in the obese individuals (P<0.05). Expression levels of the 25-hydroxylase CYP2J2 and the 1α-hydroxylase CYP27B1 were decreased by 71% (P<0.0001) and 49% (P<0.05), respectively, in SAT of the obese. CYP24A1 did not differ between lean and obese women, but the expression was increased by 79% (P<0.05) after weight loss. CONCLUSION: Obesity is characterized by a decreased expression of the 25-hydroxylase CYP2J2 and the 1α-hydroxylase CYP27B1 in SAT, whereas the catabolic CYP24A1 does not differ between lean and obese women. However, the expression of CYP24A1 is increased after weight loss. Accordingly, AT has the capacity to metabolize VD locally, and this can be dynamically altered during obesity and weight loss.

Anti-inflammatory effect of resveratrol on adipokine expression and secretion in human adipose tissue explants.

OBJECTIVE: Human obesity is closely associated with a state of chronic low-grade inflammation, which also involves the adipose tissue with enhanced production of bioactive substances (adipokines). Calorie restriction (CR) reduces adipocytokine production and improves metabolic profile in rodents. Some of these effects are mediated through activation of the sirtuin 1 (Sirt1) enzyme, and in this study, we investigate whether the natural phytoalexin, resveratrol (RSV), which is a potent Sirt1 activator, has anti-inflammatory effects in human adipose tissue explants. DESIGN: The effect of RSV on interleukin 1ß (IL1ß)-induced change of adipokine mRNA gene expression and secretion were measured in human adipose tissue explants. RESULTS: Exposure of human adipose tissue in vitro to IL1ß for 24 h increased secretion of the proinflammatory adipokines IL6, IL8 and monocyte chemoattractant protein 1 (MCP-1) 3-7.7-fold (P<0.05) and increased IL6, IL8, MCP-1, IL1ß and PAI-1 mRNA expression 1.3-7.2-fold (P<0.05) accordingly. Concomitant incubations with RSV reversed the IL1ß-stimulated secretion (16-36%) and gene expression (25-48%) of these adipokines. IL1ß reduced adiponectin mRNA expression (40%), a decrement that was reversed by RSV treatment. Similar effects were observed in differentiated human preadipocytes in primary culture, indicating that human adipocytes are a potential target for RSV effects. Finally, the effects were neutralized by sirtinol, a Sirt1 inhibitor. CONCLUSION: This study is the first to show anti-inflammatory effects of RSV on adipokine expression and secretion in human adipose tissue in vitro through the SIRT1 pathway. Thus, RSV is hypothesized to possess beneficial effects and might improve the metabolic profile in human obesity.

Reduced cannabinoid receptor 1 protein in subcutaneous adipose tissue of obese.

BACKGROUND: Cannabinoid 1 receptors are identified in various tissues involved in the internal metabolism including adipose tissue and the endocannabinoid system is claimed to be overactive in the obese state. To study the potential involvement of cannabinoid receptor 1 in the endocannabinoid system over-activity in adipose tissue in the obese state, we investigated the cannabinoid receptor 1 levels in adipose tissue from different fat depots in lean and obese humans. MATERIALS AND METHODS: The adipose tissue samples were analysed by Western blot and by RT-PCR. RESULTS: Both the gene expression and the protein of cannabinoid receptor 1 were lower in subcutaneous abdominal adipose tissue from obese subjects as compared with lean subjects (P < 0.01 and P = 0.058). Moreover, in lean subjects, the level of cannabinoid receptor 1 was significantly higher in subcutaneous adipose tissue compared with visceral adipose tissue (P < 0.05) for both gene expression and protein. The level of cannabinoid receptor 1 was similar between the two depots in obese subjects. The expression of cannabinoid receptor 1 was higher in subcutaneous gluteal adipose tissue as compared with subcutaneous abdominal adipose tissue (P < 0.05). CONCLUSION: We found in lean subjects, a robust lower level of cannabinoid receptor 1 in visceral adipose tissue compared with subcutaneous adipose tissue (both RNA and protein levels), but similar levels of cannabinoid receptor 1 between the two depots in obese subjects. Our present findings do not indicate that cannabinoid receptor 1 is directly involved in the endocannabinoid system over-activity in adipose tissue in obesity.

Expression of 11beta-hydroxysteroid dehydrogenase 1 and 2 in subcutaneous adipose tissue of lean and obese women with and without polycystic ovary syndrome.

OBJECTIVE: To investigate the expression of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 1 and 2 and hexose-6-phosphate dehydrogenase (H6PDH) mRNA in subcutaneous abdominal tissue from lean and obese women with and without polycystic ovary syndrome (PCOS), and to investigate the association between these enzymes and different measures of insulin sensitivity. DESIGN: Cross-sectional study. SUBJECTS: A total of 60 women, 36 women with PCOS, 17 lean (lean PCOS, LP) and 19 obese (obese PCOS, OP) and 24 age- and weight-matched control women, 8 lean (lean controls, LC) and 16 obese (obese controls, OC). Subcutaneous adipose tissue was collected from the abdomen. Peripheral insulin sensitivity was assessed by the euglycemic hyperinsulinemic clamp and determined as glucose disposal rate and insulin sensitivity index. Whole-body insulin sensitivity was calculated using homeostasis model assessment insulin resistance index. Body composition was evaluated by dual X-ray absorptiometry. Adipose mRNA expression of leptin and adiponectin were determined by real-time PCR. RESULTS: Polycystic ovary syndrome (P<0.05) and obesity (P<0.05) were independently associated with increased expression of 11beta-HSD1 mRNA. The subgroups LP and OC had increased 11beta-HSD1 and 11beta-HSD2 mRNA expression compared with LC (P<0.05, P<0.05). There were no effects of PCOS or obesity on11beta-HSD2 or H6PDH mRNA expression. Decreased peripheral insulin sensitivity (P<0.001) and increased upper body fat distribution (P<0.01) were associated with increased expression of 11beta-HSD1, but neither 11beta-HSD2 nor H6PDH. CONCLUSION: Polycystic ovary syndrome and obesity are independently associated with increased expression of 11beta-HSD1. This may lead to increased conversion of cortisone to cortisol in the peripheral adipose tissue and subsequently increased glucocorticoid activity. Decreased peripheral insulin sensitivity and central obesity was associated with increased expression of 11beta-HSD1.

[Cognitive training in rehabilitation: a program to treat mild cognitive impairment]. / Gedächtnismanagementtraining in der Rehabilitation (KTR): Programm zur Behandlung leichter kognitiver Beeinträchtigungen.

As part of a research project, a behavior therapy-oriented cognitive training program, designed to improve the cognitive ability of middle-aged employees (50 to 59 years), was implemented and evaluated. The goal of the training program was to analyze the cognitive, affective and behavior-related conditions of memory deficits in the workplace. The training focused on two topics: (1) development of new job tasks, (2) lack of time and deadline constraints. A total of 316 patients in the psychosomatic clinic of Bad Neustadt participated in the study. Those who showed cognitive impairments took part in the cognitive training program during the intervention phase. During the control phase, patients with cognitive impairments received no additional intervention. The neuropsychological screening was repeated with patients with cognitive impairments at discharge. The findings show that the objective and subjective memory performance of the participants improved significantly compared to that of the control group. At the 1-year follow-up, 84% of the participants reported that they could use the training strategies in their job.

Low Sirt1 expression, which is upregulated by fasting, in human adipose tissue from obese women.

OBJECTIVE: Calorie restriction increases the life span in a number of different organisms. This effect is dependent upon activation of the Sirt1 enzyme, and many of the beneficial effects of calorie restriction can be mimicked using resveratrol, which activates the Sirt1 enzyme. Nothing is known about this system in human adipose tissue; therefore, we investigated this system in human adipose tissue. DESIGN: Sirt1 mRNA was measured in adipose tissue biopsies from human volunteers before and after 6 days of total fasting. In addition, adipose tissue from lean and obese individuals was compared and in vitro investigations were performed. RESULTS: Long-term total fasting (6 days) of nine human volunteers increased Sirt1 mRNA expression in subcutaneous adipose tissue more than twofold (0.197-0.454 arbitrary units, P<0.05). Likewise, lean women (n=12) had more than twofold higher Sirt1 expression in subcutaneous adipose tissue compared to obese women (n=12; 0.33-0.73 arbitrary units, P<0.05). Sirt1 was equally expressed in the stroma-vascular fraction and the isolated adipocyte fraction. Finally, in vitro, we demonstrated that resveratrol (a Sirt1 activator) significantly enhanced the lipolytic effect of epinephrine in human adipose tissue (P<0.05). CONCLUSION: Human adipose tissue contains Sirt1 and the expression of Sirt1 can be regulated by calorie restriction as in other species. Furthermore, we demonstrated that resveratrol affects human fat-cell metabolism similar to the effects in rodents (that is, increased epinephrine induced lipolysis). These findings indicated that the beneficial effects of calorie restriction in humans might involve the activation of Sirt1. Thus, based on these findings, we propose that Sirt1 might play important roles for the beneficial effects of calorie restriction in humans.

AMP-activated protein kinase activation prevents denervation-induced decline in gastrocnemius GLUT-4.

This study was designed to determine whether the reductions in GLUT-4 seen in 3-day-denervated muscles can be prevented through chemical activation of 5'-AMP-activated protein kinase (AMPK). Muscle AMPK can be chemically activated in rats using subcutaneous injections with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). In this study, the tibial nerve was sectioned on one side; the other was sham operated but without nerve section. Acute injections of AICAR resulted in significantly increased AMPK activity in denervated gastrocnemius but not soleus muscles. Acetyl-CoA carboxylase activity, a reporter of AMPK activation, declined in both gastrocnemius and soleus in both denervated and contralateral muscles. Three days after denervation, GLUT-4 levels were significantly decreased by approximately 40% in gastrocnemius muscles and by approximately 30% in soleus muscles. When rats were injected with AICAR (1 mg/g body wt) for 3 days, the decline in GLUT-4 levels was prevented in denervated gastrocnemius muscles but not in denervated soleus muscles. The extent of denervation-induced muscle atrophy was similar in AICAR-treated vs. saline-treated rats. These studies provide evidence that some effects of denervation may be prevented by chemical activation of the appropriate signaling pathways.

Effects of thyroid state on AMP-activated protein kinase and acetyl-CoA carboxylase expression in muscle.

AMP-activated protein kinase (AMPK) consists of three subunits: alpha, beta, and gamma. Two isoforms exist for the alpha-subunit (alpha(1) and alpha(2)), two for the beta-subunit (beta(1) and beta(2)), and three for the gamma-subunit (gamma(1), gamma(2), and gamma(3)). Although the specific roles of the beta- and gamma-subunits are not well understood, the alpha-subunit isoforms contain the catalytic site and also the phosphorylation/activation site for the upstream kinase. This study was designed to determine the role of thyroid hormones in controlling expression levels of these AMPK subunits and of one downstream target, acetyl-CoA carboxylase (ACC), in muscle. AMPK subunit and ACC levels were determined by Western blots in control rats, in rats given 0.01% propylthiouracil (PTU) in drinking water for 3 wk, and in rats given 3 mg of thyroxine and 1 mg of triiodothyronine per kilogram chow for 1 or 3 wk. In gastrocnemius muscle, all isoforms of AMPK subunits were significantly increased in rats given thyroid hormones for 3 wk vs. those treated with PTU. Similar patterns were seen in individual muscle types. Expression of muscle ACC was also significantly increased in response to 3 wk of treatment with excess thyroid hormones. Muscle content of malonyl-CoA was elevated in PTU-treated rats and depressed in thyroid hormone-treated rats. These data provide evidence that skeletal muscle AMPK subunit and ACC expression is partially under the control of thyroid hormones.

Phosphorylation-activity relationships of AMPK and acetyl-CoA carboxylase in muscle.

AMP-activated protein kinase (AMPK) is activated during muscle contraction in response to the increase in AMP and decrease in phosphocreatine (PCr). Once activated, AMPK has been proposed to phosphorylate a number of targets, resulting in increases in glucose transport, fatty acid oxidation, and gene transcription. Although it has been possible to directly observe phosphorylation of one of these targets, acetyl-CoA carboxylase (ACC) in vitro, it has been more difficult to obtain direct evidence of ACC phosphorylation in contracting skeletal muscle. In these experiments using a phosphoserine antibody to ACC and a phosphothreonine antibody to AMPK, evidence was obtained for phosphorylation and activation of ACC in vitro, in gastrocnemius muscle electrically stimulated at different frequencies, and in muscle from rats running on the treadmill. Significant negative linear correlations between phospho-ACC and ACC activity were observed in all models (P < 0.01). The decline in ACC activity was related to the decrease in PCr and the rise in AMP. A relationship between phospho-AMPK (threonine 172) and activity of AMPK immunoprecipitated with anti-alpha(2) subunit antibody preparation was also observed. These data provide the first evidence of a direct link between extent of phosphorylation of these proteins at sites recognized by the antibodies and activity of the enzymes in electrically stimulated muscle and in muscle of rats running on the treadmill.

Improved results in the management of surgical candidates with lung cancer.

BACKGROUND: Perioperative mortality and morbidity after lung resection for carcinoma are generally reported to be 3% to 6% and 15% to 30%, respectively, and higher in the elderly and those with limited cardiopulmonary reserve. METHODS: To minimize this risk and extend the surgical option to more high-risk patients, we adopted a protocol in 1991 that included preoperative digitalis, subcutaneous heparin and venoocclusive stockings, aggressive perioperative pulmonary toilet, and video-directed limited resections for many patients with limited pulmonary reserve. In October 1996, we reviewed our results with 173 consecutive patients (median age, 60 years; range, 17 to 89 years) undergoing operation for suspected lung carcinoma. Forty-one patients were 70 years old or older, and 70 patients were considered high risk on the basis of advanced age (> or = 70 years), poor cardiac or pulmonary reserve, or serious medical comorbidity. Procedures included pneumonectomy (n = 31), lobectomy (n = 83), bilobectomy (n = 12), and limited resection (n = 45). Two patients had unresectable disease. RESULTS: Hospital mortality was 1.6% (3/173) and morbidity was experienced by 15% (26/173). Among the high-risk subgroup mortality was 4.2% (3/70) and morbidity was 20% (14/70; p < 0.03). For the older patients these values were 4.8% (2/41) and 17.9% (7/41), respectively. CONCLUSIONS: Morbidity and mortality from lung resections may be minimized with the perioperative management strategy outlined above. This would allow more high-risk patients to benefit from surgical resection, and do so with an acceptably low risk.

The value of immunohistochemistry of pleural biopsy specimens in the differential diagnosis between malignant mesothelioma and metastatic carcinoma.

The aim of the present study was to evaluate the aid of immunohistochemistry in the differential diagnosis between pleural malignant mesothelioma (MM) and secondary pleural carcinoma in pleural biopsy specimens. A series of MM (epithelial or biphasic type) (n = 39), and a series of secondary pleural carcinomas (n = 25) were studied with a panel of monoclonal antibodies against Ber-EP4, CEA, and Leu-MI (CD-15). Considering the markers one, two or three at a time, computer analysis was conducted to identify the immunophenotype with the highest sensitivity and specificity for both MM (n = 39) and adenocarcinoma (n = 19). The CEA/CD-15 marker combination had the highest specificity (MM = 100%; adenocarcinoma = 82.1%), and sensitivity (MM = 82.1%; adenocarcinoma = 100%) considering both categories of tumours. Positive reaction for CEA and/or CD-15 was the best indicator of metastatic adenocarcinoma. Negative reaction for CEA/CD-15 and Ber-EP4/CD-15 showed up to be 100% specific for MM. Positive reaction for one, two or even all these antigens, however, occurred in nine tumours predetermined as MM according to their gross appearance, and absence of other primary malignancies at autopsy. Ultrastructural examination (n = 7) of these tumours strongly supported the diagnosis in one case, and did by no means exclude the diagnosis MM in the other six cases. Neither did any of the tumours reveal ultrastructural signs of adenomatous differentiation, and they did not express PAS/diastase resistant globules, amylase or surfactant. Anti -Ber-EP4 was the antibody most frequently expressed in MM (n = 8). Until antibodies specific for MM are available, the limitation of immunohistochemistry to solve the diagnosis in all cases of pleural malignant tumours has to be considered.

Analysis of the effects of deep mechanical massage in the porcine model.

Deep mechanical massage has been advocated as an alternative or adjunctive therapy for the contouring of subcutaneous fat and as a treatment for cellulite. We evaluated the effects of deep mechanical massage using two pig models. Yucatan pigs were divided into three groups (n = 4). One side of each body received 4, 10, or 20 treatments and the other side served as a control. Full-thickness tissue sections, including the underlying muscle, were harvested from identical treated and untreated regions. Examination of these regionally matched samples revealed an accumulation of dense, longitudinal collagen bands in the middle dermal and deep subdermal regions, which progressively increased with the number of treatments. Distortion and disruption of adipocytes was noted. In Yorkshire pigs, force-transducing balloon catheters were surgically placed between the deep subcutaneous tissue and muscle fascia. Catheters were inserted into two regions with different skin and subcutaneous tissue characteristics, the midflank and the hip. Standardized maneuvers were performed at suction settings 3, 5, 7, and 9 to record baseline tissue forces. Each maneuver carried a unique force signature. The measurement of tissue forces was repeated on the opposite side after 10 standardized treatment sessions. Analysis showed a significant reduction of measured forces at the midflank after the treatments. The actual force measured with each particular maneuver varied between different operators but not with different suction settings, suggesting that the technique of administering the treatments is the primary factor in creating the force within the tissue. This leads to the conclusion that deep mechanical massage is highly dependent on the individual operator of the device.

Immunohistochemical and electron microscopic study of twelve adenomatoid tumors.

Twelve adenomatoid tumors were examined immunohistochemically with antibody probes to keratin and factor VIII-related antigen (FVIII-RA). None of the tumors labeled with FVIII-RA antibodies, whereas all but one labeled for keratin. Electron microscopy was done on tissue from paraffin-embedded blocks from all but one of the adenomatoid tumors. Both the immunohistochemical and electron microscopic results favor a mesothelial origin of adenomatoid tumors.

Titanium tetrachloride: an unusual agent with the potential to create severe burns.

Titanium tetrachloride (TiCl4), an intermediate compound in the production of white pigment, can cause severe burns. Two cases are reported in which TiCl4 created 18% to 20% total body surface area burns. These full-thickness injuries were the combined consequence of hydrochloric acid and the heat that was generated in areas where this otherwise stable compound was mixed with perspiration. TiCl4 combined with water is extremely dangerous, and its immediate treatment--towel drying before irrigation--makes it unique among chemicals. Our experience suggests that in most cases grafting will be required. These chemical burns were self-limited and had no notable systemic sequelae. Wound biopsy specimens taken on postburn days 3 and 6 were subjected to immunostaining that showed that TiCl4 did not retard wound healing. Exposure time to TiCl4 vapor will determine the pulmonary and ophthalmologic involvement in each case. Clinical awareness of the propensity of TiCl4 to react with water--even when that water is in the form of perspiration--is vital because prompt management can limit the extent of injury.

[Thyroid cancer in the county of North Jutland. Diagnosis, treatment and prognosis]. / Cancer thyreoideae i Nordjyllands Amt. Diagnostik, behandling og prognose.

Data concerning 153 patients diagnosed with thyroid cancer during the 15-year period 1976-90 were retrospectively analyzed. Fine needle aspiration was performed in 98 patients. False negative smears occurred in 16.5% of the aspirates, most frequently in the papillary carcinoma group in which 20.5% of the smears contained cystic material. It is conducted, that smears containing cystic material without follicular epithelial cells should not be considered adequate for diagnosis and should always be repeated. The tumors were histologically re-examined. In 14 cases the histological diagnosis was re-classified. The ENT-department of Aalborg Hospital treated 136 of the patients. Unilateral lobectomy was performed in localised, unilateral cases of papillary carcinoma and as a main rule, total thyreoidectomy in all other cases of differentiated carcinoma and in all cases of medullary carcinoma. There were few postoperative complications. Unilateral recurrent nerve paralysis occurred in two percent of the patients. The prognosis of differentiated and medullary carcinoma was good and in accordance with other studies in which total thyreoidectomy has been performed in all cases. The pTNM classification did not correlate well with the actuarial survival.

Effect of weight loss and exercise on angiogenic factors in the circulation and in adipose tissue in obese subjects.

BACKGROUND: Vascular growth is a prerequisite for adipose tissue (AT) development and expansion. Some AT cytokines and hormones have effects on vascular development, like vascular endothelial growth factor (VEGF-A), angiopoietin (ANG-1), ANG-2 and angiopoietin-like protein-4 (ANGPTL-4). METHODS: In this study, the independent and combined effects of diet-induced weight loss and exercise on AT gene expression and proteins levels of those angiogenic factors were investigated. Seventy-nine obese males and females were randomized to: 1. Exercise-only (EXO; 12-weeks exercise without diet-restriction), 2. Hypocaloric diet (DIO; 8-weeks very low energy diet (VLED) + 4-weeks weight maintenance diet) and 3. Hypocaloric diet and exercise (DEX; 8-weeks VLED + 4-weeks weight maintenance diet combined with exercise throughout the 12 weeks). Blood samples and fat biopsies were taken before and after the intervention. RESULTS: Weight loss was 3.5 kg in the EXO group and 12.3 kg in the DIO and DEX groups. VEGF-A protein was non-significantly reduced in the weight loss groups. ANG-1 protein levels were significantly reduced 22-25% after all three interventions (P < 0.01). The ANG-1/ANG-2 ratio was also decreased in all three groups (P < 0.05) by 27-38%. ANGPTL-4 was increased in the EXO group (15%, P < 0.05) and 9% (P < 0.05) in the DIO group. VEGF-A, ANG-1, and ANGPTL-4 were all expressed in human AT, but only ANGPTL-4 was influenced by the interventions. CONCLUSIONS: Our data show that serum VEGF-A, ANG-1, ANG-2, and ANGPTL-4 levels are influenced by weight changes, indicating the involvement of these factors in the obese state. Moreover, it was found that weight loss generally was associated with a reduced angiogenic activity in the circulation.