Amphetamine increases vascular permeability by modulating endothelial actin cytoskeleton and NO synthase via PAR-1 and VEGF-R.

Abuse of amphetamine-type stimulants is linked to cardiovascular adverse effects like arrhythmias, accelerated atherosclerosis, acute coronary syndromes and sudden cardiac death. Excessive catecholamine release following amphetamine use causes vasoconstriction and vasospasms, over time leading to hypertension, endothelial dysfunction or even cardiotoxicity. However, immediate vascular pathomechanisms related to amphetamine exposure, especially endothelial function, remain incompletely understood and were analyzed in this study. Pharmaco-pathological effects of acute d-amphetamine-sulfate (DAM) were investigated ex vivo using contraction-force measurements of rat carotid artery rings and in vitro using label-free, real-time electrochemical impedance spectroscopy (EIS) on endothelial and smooth muscle cells. Specific receptor and target blocking was used to identify molecular targets and to characterize intracellular signaling. DAM induced vasodilation represented by 29.3±2.5% decrease in vascular tone (p<0.001) involving vascular endothelial growth factor receptor (VEGF-R) and protease activated receptor 1 (PAR-1). EIS revealed that DAM induces endothelial barrier disruption (-75.9±1.1% of initial cellular impedance, p<0.001) also involving VEGF-R and PAR-1. Further, in response to DAM, Rho-associated protein kinase (ROCK) mediated reversible contraction of actin cytoskeleton resulting in endothelial barrier disruption. Dephosphorylation of Serine1177 (-50.8±3.7%, p<0.001) and Threonine495 (-44.8±6.5%, p=0.0103) of the endothelial NO synthase (eNOS) were also observed. Blocking of VEGF-R and PAR-1 restored baseline eNOS Threonine495 phosphorylation. DAM induced vasodilation, enhanced vascular permeability and actin cytoskeleton contraction and induced eNOS hypophosphorylation involving VEGF-R, PAR-1 and ROCK. These results may contribute to a better understanding of severe adverse cardiovascular effects in amphetamine abuse.

Age-related changes in the midpalatal suture: Comparison between CBCT staging and bone micromorphology.

The age-related maturation of the human midpalatal suture is challenging to predict, but critical for successful non-surgical rapid maxillary expansion (RME). While cone-beam computed tomography (CBCT) can be used to categorize the suture into stages, it remains unclear how well the stages predict the actual micromorphology of the palate. To address this clinically relevant question, we used CBCT together with three-dimensional micro-computed tomography (µCT) analysis on 24 human palate specimens from individuals aged 14-34 years. We first classified the specimens into stages (A-E) using CBCT images and then correlated the results with our comprehensive µCT analysis. Our analysis focused on several factors, including bone volume fraction (BV/TV), sutural width, volume, interdigitation, ossification, and their associations with age, CBCT stage, and sex. Our µCT analysis revealed a decrease in sutural width and volume after the age of 20 years, accompanied by sutural closure beginning in the palatal segment. The overall rate of ossification remained low but increased after the age of 20 years. No significant differences were found between males and females. Importantly, we also found no correlation between individual age and CBCT stages. Furthermore, there was no association between CBCT stages and patalal suture volume, ossification and interdigitation. Taken together, our findings cast doubt on the reliability of CBCT stage as a means of predicting skeletal maturity of the palatal suture, as it appears to lack the precision required to accurately assess the true micromorphology of the palatal suture. Future investigations should explore whether alternative CBCT parameters may be more useful in addressing the challenging question of whether RME requires surgical bone weakening.

Mechanical-induced bone remodeling does not depend on Piezo1 in dentoalveolar hard tissue.

Mechanosensory ion channels are proteins that are sensitive to mechanical forces. They are found in tissues throughout the body and play an important role in bone remodeling by sensing changes in mechanical stress and transmitting signals to bone-forming cells. Orthodontic tooth movement (OTM) is a prime example of mechanically induced bone remodeling. However, the cell-specific role of the ion channels Piezo1 and Piezo2 in OTM has not been investigated yet. Here we first identify the expression of PIEZO1/2 in the dentoalveolar hard tissues. Results showed that PIEZO1 was expressed in odontoblasts, osteoblasts, and osteocytes, while PIEZO2 was localized in odontoblasts and cementoblasts. We therefore used a Piezo1floxed/floxed mouse model in combination with Dmp1cre to inactivate Piezo1 in mature osteoblasts/cementoblasts, osteocytes/cementocytes, and odontoblasts. Inactivation of Piezo1 in these cells did not affect the overall morphology of the skull but caused significant bone loss in the craniofacial skeleton. Histological analysis revealed a significantly increased number of osteoclasts in Piezo1floxed/floxed;Dmp1cre mice, while osteoblasts were not affected. Despite this increased number of osteoclasts, orthodontic tooth movement was not altered in these mice. Our results suggest that despite Piezo1 being crucial for osteoclast function, it may be dispensable for mechanical sensing of bone remodeling.

A previously uncharacterized Factor Associated with Metabolism and Energy (FAME/C14orf105/CCDC198/1700011H14Rik) is related to evolutionary adaptation, energy balance, and kidney physiology.

In this study we use comparative genomics to uncover a gene with uncharacterized function (1700011H14Rik/C14orf105/CCDC198), which we hereby name FAME (Factor Associated with Metabolism and Energy). We observe that FAME shows an unusually high evolutionary divergence in birds and mammals. Through the comparison of single nucleotide polymorphisms, we identify gene flow of FAME from Neandertals into modern humans. We conduct knockout experiments on animals and observe altered body weight and decreased energy expenditure in Fame knockout animals, corresponding to genome-wide association studies linking FAME with higher body mass index in humans. Gene expression and subcellular localization analyses reveal that FAME is a membrane-bound protein enriched in the kidneys. Although the gene knockout results in structurally normal kidneys, we detect higher albumin in urine and lowered ferritin in the blood. Through experimental validation, we confirm interactions between FAME and ferritin and show co-localization in vesicular and plasma membranes.

Early enzyme replacement therapy prevents dental and craniofacial abnormalities in a mouse model of mucopolysaccharidosis type VI.

Mucopolysaccharidosis VI (MPS VI) is a hereditary lysosomal storage disease caused by the absence of the enzyme arylsulfatase B (ARSB). Craniofacial defects are common in MPS VI patients and manifest as abnormalities of the facial bones, teeth, and temporomandibular joints. Although enzyme replacement therapy (ERT) is the treatment of choice for MPS VI, the effects on the craniofacial and dental structures are still poorly understood. In this study, we used an Arsb-deficient mouse model (Arsb m/m ) that mimics MPS VI to investigate the effects of ERT on dental and craniofacial structures and compared these results with clinical and radiological observations from three MPS VI patients. Using micro-computed tomography, we found that the craniofacial phenotype of the Arsb m/m mice was characterized by bone exostoses at the insertion points of the masseter muscles and an overall increased volume of the jaw bone. An early start of ERT (at 4 weeks of age for 20 weeks) resulted in a moderate improvement of these jaw anomalies, while a late start of ERT (at 12 weeks of age for 12 weeks) showed no effect on the craniofacial skeleton. While teeth typically developed in Arsb m/m mice, we observed a pronounced loss of tooth-bearing alveolar bone. This alveolar bone loss, which has not been described before in MPS VI, was also observed in one of the MPS VI patients. Interestingly, only an early start of ERT led to a complete normalization of the alveolar bone in Arsb m/m mice. The temporomandibular joints in Arsb m/m mice were deformed and had a porous articular surface. Histological analysis revealed a loss of physiological cartilage layering, which was also reflected in an altered proteoglycan content in the cartilage of Arsb m/m mice. These abnormalities could only be partially corrected by an early start of ERT. In conclusion, our results show that an early start of ERT in Arsb m/m mice achieves the best therapeutic effects for tooth, bone, and temporomandibular joint development. As the MPS VI mouse model in this study resembles the clinical findings in MPS VI patients, our results suggest enzyme replacement therapy should be started as early as possible.

Arginine metabolism and nitric oxide turnover in the ZSF1 animal model for heart failure with preserved ejection fraction.

Endothelial dysfunction and altered nitric oxide (NO) metabolism are considered causal factors in heart failure with preserved ejection fraction (HFpEF). NO synthase activity depends on the availability of arginine and its derivatives. Thus, we analyzed arginine, associated metabolites, arginine-metabolizing enzymes and NO turnover in 20-week-old female healthy lean (L-ZSF1) and obese ZSF1 rats (O-ZSF1) with HFpEF. Serum, urine and lysates of liver, kidney and heart were analyzed. There were significantly lower lysine (- 28%), arginine (- 31%), homoarginine (- 72%) and nitrite (- 32%) levels in serum of O-ZSF1 rats. Ornithine (+ 60%) and citrulline (+ 20%) levels were higher. Similar results were found in the heart. Expression of arginine consuming enzymes in liver and kidney was unchanged. Instead, we observed a 5.8-fold higher arginase 1 expression, presumably of granulocyte origin, in serum and > fourfold increased cardiac macrophage invasion in O-ZSF1. We conclude that inflammatory cells in blood and heart consume arginine and probably homoarginine via arginase 1 and inducible NO synthase and release ornithine and citrulline. In combination with evidence for decreased NO turnover in O-ZSF1 rats, we assume lower arginine bioavailability to endothelial NO synthase.

Structural and functional alterations in heart and skeletal muscle following severe TAC in mice: impact of moderate exercise training.

BACKGROUND: Heart failure (HF) is the leading cause of death in western countries. Cardiac dysfunction is accompanied by skeletal alterations resulting in muscle weakness and fatigue. Exercise is an accepted interventional approach correcting cardiac and skeletal dysfunction, thereby improving mortality, re-hospitalization and quality of life. Animal models are used to characterize underpinning mechanisms. Transverse aortic constriction (TAC) results in cardiac pressure overload and finally HF. Whether exercise training improves cardiac remodeling and peripheral cachexia in the TAC mouse model was not analyzed yet. In this study, 2 weeks post TAC animals were randomized into two groups either performing a moderate exercise program (five times per week at 60% VO2 max for 40 min for a total of 8 weeks) or staying sedentary. RESULTS: In both TAC groups HF characteristics reduced ejection fraction (- 15% compared to sham, p < 0.001), cardiac remodeling (+ 22.5% cardiomyocyte cross sectional area compared to sham; p < 0.001) and coronary artery congestion (+ 34% diameter compared to sham; p = 0.008) were observed. Unexpectedly, peripheral cachexia was not detected. Furthermore, compared to sedentary group animals from the exercise group showed aggravated HF symptoms [heart area + 9% (p = 0.026), heart circumference + 7% (p = 0.002), right ventricular wall thickness - 30% (p = 0.003)] while muscle parameters were unchanged [Musculus soleus fiber diameter (p = 0.55), Musculus extensor digitorum longus contraction force (p = 0.90)]. CONCLUSION: The severe TAC model is inappropriate to study moderate exercise effects in HF with respect to cardiac and skeletal muscle improvements. Further, the phenotype induced by different TAC procedures should be well documented and taken into account when planning experiments.

Structural and functional alterations in heart and skeletal muscle following severe TAC in mice: impact of moderate exercise training

BACKGROUND: Heart failure (HF) is the leading cause of death in western countries. Cardiac dysfunction is accompanied by skeletal alterations resulting in muscle weakness and fatigue. Exercise is an accepted interventional approach correcting cardiac and skeletal dysfunction, thereby improving mortality, re-hospitalization and quality of life. Animal models are used to characterize underpinning mechanisms. Transverse aortic constriction (TAC) results in cardiac pressure overload and finally HF. Whether exercise training improves cardiac remodeling and peripheral cachexia in the TAC mouse model was not analyzed yet. In this study, 2 weeks post TAC animals were randomized into two groups either performing a moderate exercise program (five times per week at 60% VO2 max for 40 min for a total of 8 weeks) or staying sedentary. RESULTS: In both TAC groups HF characteristics reduced ejection fraction (- 15% compared to sham, p < 0.001), cardiac remodeling (+ 22.5% cardiomyocyte cross sectional area compared to sham; p < 0.001) and coronary artery congestion (+ 34% diameter compared to sham; p = 0.008) were observed. Unexpectedly, peripheral cachexia was not detected. Furthermore, compared to sedentary group animals from the exercise group showed aggravated HF symptoms [heart area + 9% (p = 0.026), heart circumference + 7% (p = 0.002), right ventricular wall thickness - 30% (p = 0.003)] while muscle parameters were unchanged [Musculus soleus fiber diameter (p = 0.55), Musculus extensor digitorum longus contraction force (p = 0.90)]. CONCLUSION: The severe TAC model is inappropriate to study moderate exercise effects in HF with respect to cardiac and skeletal muscle improvements. Further, the phenotype induced by different TAC procedures should be well documented and taken into account when planning experiments.