bluuebunny/crossref_metadata_2025_split huggingface dataset:
Involvement of Lysyl Oxidase in the Pathogenesis of Arterial Stiffness in Chronic Kidney Disease
Authors: Sharma Ravindra K. , Krishnan Suraj , Jakkidi Nishka , Gomes Joshua , Li Shiyu K. , Mohandas Rajesh 
Published: May 2022

RationaleIncreased vascular stiffness is a key mediator of cardiovascular disease (CVD) in patients with chronic kidney disease (CKD). Increased vascular stiffness in CKD is thought to be the consequence of long‐standing hypertension and vascular calcification. However, vascular stiffness is increased early in patients with CKD, even before metabolic complications develop. Lysyl Oxidase (LOX) is a key mediator of collagen crosslinking and matrix remodeling. Up‐regulation of LOX has been associated with increased tissue stiffness in cancer and wound healing. We hypothesize that increased LOX might be a key mechanism for increased vascular stiffness in CKD. β‐aminopropionitrile (BAPN), a LOX inhibitor, was used to test the hypothesis.Methods5/6 nephrectomy (Nx) or Sham surgery was performed in 8‐weeks old male C57BL/6 mice. 2‐weeks after Nx, mice were randomized to BAPN (300 mg/kg/day in water) or vehicle for 4‐weeks. Vascular stiffness was assessed by pulse wave velocity (PWV). LOX levels were measured by ELISA and qPCR. Collagen crosslinking was measured by mass spectrometry. Comparisons between groups were made using one‐way ANOVA, p‐Value <0.05 was accepted as significant.ResultsNx resulted in decreased renal function at 8 weeks but no change in blood pressure or calcification. (~2‐3‐fold increase in creatinine, urea, and cystatin C) compared to control mice. Nx surgery resulted in increase in PWV (Con:179±32, Nx: 306 ± 47 cm/s, p<0.001). This was associated with an increased LOX in aorta (Con:118±8, Nx: 311±13 pg/mg dry weight, p<0.001). Mice with Nx surgery had an increased total collagen contents (183%, p<0.001) as well as cross‐linked collagen (DPD; 83%, p<0.05, PYD: 270% p<0.01). BAPN treatment significantly attenuates PWV (211± 49 cm/s, p<0.01), LOX (256±21 pg/mg dry weight, p<0.05) and total collagen contents (23%, p<0.01). BAPN treatment reduced the crosslinked collagens (DPD and PYD) but this did not reach statistical significance.ConclusionOur findings suggest: a) Increased vascular stiffness in CKD is independent of BP and calcification b) LOX mediated changes in extracellular matrix are at least in part responsible for the pathogenesis of arterial stiffness in CKD. Inhibition of excessive LOX may have therapeutic potential in alleviating increased vascular stiffness in CKD.
Does Macrophage Stimulation by CCL5 Alter Melanoma Migratory Behavior?
Authors: Meyer Jacob T. , Baer Robert W. 
Published: May 2022

BackgroundCCL5 (RANTES, regulated upon activation, normal T‐cell expressed and secreted) is secreted not only by T‐cells, but also by melanoma cells into the tumor microenvironment. The current project is part of a bigger effort by our laboratory to determine how the stimuli present in the tumor microenvironment may contribute to the progression of melanoma. Here, we explore CCL5 receptor expression on monocytes, macrophages, and melanoma cells, and whether endogenous or exogenous CCL5 stimulation of THP‐1 macrophages produce phenotypical changes that affect melanoma migratory behavior.MethodsWe used WM278 and LM‐Mel‐45 melanoma cells and THP‐1 monocytes before and after conversion to macrophages by 2 days exposure to 50 ng/ml PMA. To examine expression of the 3 key CCL5 receptors, we compared median fluorescence intensity (MFI) of antibody‐exposed cells to MFI of unlabeled cells using antibodies for CCR1, CCR3, and CCR5. We gated out debris and multiples, corrected for spillover, and analyzed the samples on a Bio‐Rad S3e cell sorter. We used recombinant CCL5 (100 ng/ml), the CCR1 & CCR3 receptor antagonist, UCB35625 (100 nM) and the CCR5 antagonist, maraviroc (100 ng/ml). Melanoma migration rate was measured by a 16 h wound‐closure assay. Treatments included: Control, CCL5 only, maraviroc only, UCB35625 only, maraviroc and UCB35625 together, and CCL5 plus both blockers. Wound closure of LM‐Mel‐45 melanoma alone, a 1:4 mixture of macrophages to melanoma, or LM‐Mel‐45 melanoma treated with macrophage‐conditioned medium were compared in each treatment condition.ResultsCCL5 receptor expression was not found on WM278 melanoma cells. LM‐Mel‐45 melanoma express CCR1 but not CCR3 or CCR5. CCR1 and CCR5, but not CCR3, are expressed by both THP‐1 monocytes and THP‐1 macrophages. Melanoma cells alone or when co‐cultured with previously treated macrophages migrated at 14.3 ± 0.4 μm/h, but neither CCL5 stimulation nor receptor blockade changed migration significantly. When LM‐Mel‐45 melanoma cells were treated with conditioned macrophage medium, velocity increased to 16.8 ± 0.7 μm/h.ConclusionsThis work suggests that CCL5 does not directly impact melanoma migration either by endogenous expression or by exogenous addition. Even in the presence of CCL5 or antagonist‐exposed macrophages, we found no phenotypical changes that altered the physical interactions of macrophages on melanoma migratory rate. There is a small but significant increase in velocity of melanoma cells when treated with conditioned macrophage medium.
Bilateral Nephrectomy Impairs Cerebral Oxygen Delivery After Acute Hemodilution Anemia in Rats
Authors: Chin Kyle , Steinberg Benjamin E. , Goldenberg Neil M. , Baker Andrew J. , Mazer C. D. , Hare Gregory M. T. 
Published: May 2022

HypothesisWe hypothesize that renal sensing of hypoxia is essential for activation of cardiovascular responses needed to maintain cerebral oxygen delivery during acute anemia and that bilateral nephrectomy will negatively affect brain oxygen delivery during acute hemodilutional anemia.MethodsAll experiments were approved by the Animal Care and Use Committee at St. Michael’s Hospital and conducted in accordance with ARRIVE‐2 guidelines. Sham or bilateral nephrectomy (n=5) were performed on isoflurane anesthetized Sprague‐Dawley rats prior to performing acute hemodilution (50% estimated blood volume) 1:1 with 6% hydroxyethyl starch. Heart rate (HR), mean arterial pressure (MAP) and rectal temperature were monitored continuously. Arterial blood gases and cooximetry samples were measured before and after hemodilution. Continuous measurements of brain microvascular oxygen tension (PbrO2) were measured via a phosphorescence quenching method using an intravascular oxygen probe, Oxyphor PdG4, and an OxyLED phosphorometer. Data were tested for normality and analysis performed by two‐way ANOVA with p<0.05 taken to be significant.ResultsBilateral nephrectomy resulted in a reduction in baseline HR (p<0.004). Hemodilution resulted in a comparable reduction in hemoglobin concentration in both groups (Sham: 5.1±0.3 g/dL; Nephrectomy: 4.2±0.4 g/dL) with no differences in arterial PaO2 or PaCO2. After hemodilution, HR increased significantly in both groups (p<0.05). MAP tended to be lower in the bilateral nephrectomy group and decreased transiently in both groups following hemodilution (p<0.01). Brain microvascular pO2 (PbrO2)was not significantly different between nephrectomy and sham at baseline. Post‐hemodilution the PbrO2 was significantly lower in nephrectomised rats (11.5±5.1 mmHg) compared to sham operated rats (25.4±11.3 mmHg) (p=0.031).ConclusionsWe observed that bilateral nephrectomy resulted in changes in baseline physiology, including a lower HR. Hemodilution resulted in a comparable reduction in hemoglobin concentration, without difference in PaO2 or PaCO2. MAP decreased more profoundly immediately after hemodilution in nephrectomised animals, and recovered with time. Nephrectomy resulted in a significant drop in brain microvascular pO2 (PbrO2) immediately after hemodilution. This was not observed in sham controls. These data suggest that the kidney serves an important sensing role during acute anemia which supports cardiovascular responses and preserves the adequacy of brain tissue oxygen delivery. Hypoxia sensing by the kidney, and other integrative systemic mechanisms, may be central to these findings.
Impaired Proteostasis, not Protein Synthesis, Limits Recovery of Aged Skeletal Muscle After Disuse Atrophy
Authors: Fuqua Jordan D. , Lawrence Marcus M. , Hettinger Zachary , Borowik Agnieszka K. , Peelor Frederick F. , Confides Amy L. , Dupont‐Versteegden Esther E. , Miller Benjamin F. 
Published: May 2022

Older individuals undergo more frequent inpatient hospitalizations and bedrest compared to younger persons, causing rapid losses of muscle mass and strength. Evidence suggests that only aged muscle fails to completely recover following disuse despite aged and adult muscle having similar levels of muscle loss. Current efforts to improve muscle recovery in older individuals are commonly aimed at increasing myofibrillar protein synthesis via mTOR stimulation despite recent evidence demonstrating that old muscle has chronically elevated levels of mTORC1 activity. We hypothesized that old muscle fails to fully recover muscle mass due to impaired proteostasis and not due limitations in protein synthesis.MethodsAdult (10 month) and old (30 month) F344BN rats were hindlimb unloaded (HU) for 14‐days to induce atrophy, followed by subsequent reloading (RE) to study muscle regrowth. During RE the rats were labeled with deuterium oxide (D2O) for 7, 15, 30, 45 or 60 days (n=4‐6 per timepoint per group) to determine bulk and individual protein synthesis and RNA synthesis. We also assessed muscle mass, fiber size by immunohistochemistry, mTORC1 signaling by western blot, and aggregate formation by tissue fractionation.ResultsAdult and old gastrocnemius (GA) muscle had significant losses of muscle mass (359 mg ±55 and 724 mg ±60 loss respectively,p<0.05) and fiber size (1296 um2±202 and 1480 um2 ±142 loss respectively, p<0.01) with HU. While adult muscle fully recovered GA mass and fiber size by day 15, old GA muscle did not fully recover during the entire 60 days of RE (18% CSA and 11% mass loss still present). Despite limited mass and fiber size recovery in old muscle, GA myofibrillar protein synthesis rates (1.45 ±0.18 vs 2.28 ±0.24, FSR%, p<0.05) and mTORC1‐related signaling were higher in old muscle compared to adult. Additionally, GA RNA synthesis rates (2.79 ±0.139 vs 5.18 ±0.684, p<0.05) and RNA concentration (183 ng ±3.6 vs 249 ng ±13.9, p<0.05), which are markers of translational capacity, were also elevated in old muscle relative to adult during RE. Old muscle had higher levels of insoluble protein aggregates, a marker of impaired proteostasis, during RE compared to adult (1.2 ±0.09 vs 1.5 ±012 fold increase, p<0.05). Lastly, we assessed individual protein synthesis rates of the whole muscle proteome and discovered that old GA muscle had a larger number of proteins with increased synthesis rates compared to adult. In conclusion, these data strongly suggest that limitations in old muscle to recover after disuse are not due to limitations in protein synthesis but are instead due to impaired proteostasis with age. Therefore, understanding how proteostasis responds during these periods surrounding unloading in old muscle are critical to improve muscle recovery after disuse.
The Ying and the Yang: Compensatory UPR Signaling Responses Observed In An In‐Vitro Model Expressing Clinical Mutant Surfactant Protein C Isoforms
Authors: Dimopoulos Thalia , Iyer Swati , Rodriguez Luis R. , Murthy Aditi , Katzen Jeremy , Beers Michael F. 
Published: May 2022

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease characterized by alveolar type II (AT2) cell dysfunction. Mutations in surfactant protein C (SP‐C) are a high effect size, recognized etiological cause of IPF. The SP‐C proprotein contains an ~100 KDa Bri2 chondromodulin‐1 and prosurfactant protein C (BRICHOS) domain that acts as a molecular chaperone preventing protein aggregation. The presence of misfolded proteins in the ER can be sensed by three transmembrane sentinel proteins – activating transcription factor 6 (ATF6), protein kinase R‐like ER kinase (PERK), and inositol‐requiring enzyme 1α (IRE1α) – which trigger an intracellular signaling pathway called the unfolded protein response (UPR). Previous characterization of our in‐vitro and in‐vivo models has demonstrated that BRICHOS mutants activate all 3 arms of the UPR, activate a proinflammatory cascade, and drive AT2 cell death. In‐vivo, their expression initiates pathological fibroproliferative lung remodeling, characteristic of human IPF. Using our in‐vitro models of BRICHOS‐driven ER stress, we explore potential IPF therapeutic intervention targeting two upstream sensors of the UPR, PERK and IRE‐1.Mouse lung epithelial (MLE‐12) cells were transfected with wildtype (WT) or BRICHOS mutant (C121G) SP‐C plasmids in media containing inhibitors of either PERK signaling or IRE‐1 RNase activity. Pathway readouts included: quantification of cell death via LDH cytotoxicity and flow cytometry, protein immunoblots to detect UPR activation and apoptotic signaling, QPCR of spliced X‐Box Binding Protein 1 (XBP1) and UPR transcriptional targets, and high‐resolution microscopy to observe altered SP‐C trafficking.Within 48 hours of transfection of MLE‐12 cells with SP‐CC121G we observed activation of the 3 arms of the UPR, upregulated downstream targets, and initiation of cell death. Inhibition of PERK resulted in down‐regulation of PERK pathway proteins and transcriptional targets, increased SP‐C protein expression, and promoted increased XBP1 splicing. Inhibition of IRE1 RNase inhibited splicing of XBP1, reduced expression of XBP1 transcriptional targets, and increased JNK phosphorylation. Inhibition of either pathway alone did not reduce cell death, though partial reduction of cell death was noted with either small molecule targeted caspase 3/8 or necroptosis inhibition.Our data suggests that while intervention directed at one arm of the UPR may block proinflammatory signaling or transcriptomic reprogramming, this strategy may not be sufficient to completely inhibit cell death. These findings highlight the interconnectivity and redundancy of the cell quality control machinery and the emerging role of necroptosis due to chronic UPR stress‐mediated cell death. Further characterization of this pathway is needed to improve IPF therapeutic outcomes.
Recovery of hyperoxia‐induced DDAH1 suppression with FXR agonist GW4064 in human pulmonary microvascular endothelial cells
Authors: Trittmann Jennifer K. , Almazroue Hanadi , Nelin Leif D. 
Published: May 2022

ObjectivePulmonary hypertension (PH) predicts mortality in patients with bronchopulmonary dysplasia (BPD), and chronic hyperoxia exposure contributes to BPD‐PH disease progression. BPD‐PH is characterized by aberrant pulmonary vascular wall proliferation/remodeling, which occludes the microvascular lumen, increasing pulmonary vascular resistance. Endogenous nitric oxide (NO) production by pulmonary vascular endothelial cells promotes apoptosis of pulmonary vascular endothelial and smooth muscle cells. In vitro studies of human pulmonary microvascular endothelial cells (hPMVEC) have shown that dimethylarginine dimethylaminohydrolase‐1 (DDAH1) promotes NO‐mediated apoptosis. DDAH1 metabolizes the majority of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of endothelial NO synthase. Therefore, our hypothesis is that pharmacologically enhancing DDAH1 expression in hPMVEC will restore endogenous endothelial NO production.MethodshPMVEC were grown at 37 ̊C to ~70% confluence. Farnesoid X receptor (FXR) agonist, GW4064 (1.25 µM) was used to bind FXR element (FXRE) of the DDAH1 promoter to enhance transcription. hPMVEC were pre‐treated with FXR agonist for 24 hours and then exposed to 85%O2 or 21%O2 for an additional 24 hours. DDAH1 protein expression was analyzed by Western blot. Groups were compared by 1‐way ANOVA.ResultsIn hPMVEC, hyperoxia exposure resulted in lower DDAH1 protein levels compared to hPMVEC grown in normoxia (N=6 in each group, p=0.003). Treatment with GW4064 prior to exposing to hyperoxia resulted in 4.3‐fold greater DDAH1 protein levels than in vehicle/85%O2 hPMVEC (N=6, p=0.044), and restored DDAH1 protein levels to 72% of that seen in the vehicle/21%O2exposed hPMVEC.ConclusionsChronic hyperoxia suppresses DDAH1 levels, and FXR agonist promotes DDAH1 recovery in hPMVEC. We speculate that promoting endogenous DDAH1 expression may prevent aberrant proliferation in the pulmonary vascular wall and thereby attenuate or prevent the vascular remodeling characteristic of pulmonary hypertension in BPD.
The Cardiorespiratory Coupling as a Background for Blood Pressure Decrease
Authors: Medviedieva Anastasiia , Shekh Vira , Iduh Mirabel D. 
Published: May 2022

The role of cardiorespiratory coupling cannot be overemphasized. Cardiorespiratory coupling has many potential benefits in maintaining homeostasis. One of manifestation of cardiorespiratory coupling is respiratory sinus arrhythmia observed at respiratory rate 6 breaths/ minute. Multiple factors, including mechanical coupling, are responsible for the fluctuation of HR and BP. However, the fine mechanisms of the autonomic, cardiovascular, and respiratory interactions till now remains unclear.A total of 156 international students were recruited from the V.N. Karazin Kharkiv National University. The sitting position was taken as a baseline according to requirements of the spirometry system. The ECG was continuously recorded at the rest and during slow breathing at 6 breaths/minute. The respiratory rate (RR) and tidal volume (TV) were recorded at the end of each stage for one minute using SPIROCOM pc based systems (XAI‐Medica, Ukraine). Systolic and diastolic blood pressure (SBP and DBP) were recorded by means automatic digital sphygmomanometer (OMRON EVOLV, Japan). The low and high frequency heart rate variability (LF and HF HRV) power were computed with CardioLab CS by fast Fourier transforming. The repeated measures MANOVA with Bonferroni correction (SPSS 22) was used to test for effects of slow breathing on HR, SBP, DBP, LnLF, LnHF, tidal volume (TV), minute ventilation (MV), and RR variables. Predictors of HR, SBP, DBP, TV, MV., and RR to slow breathing were determined using stepwise multiple linear regression analysis. Written informed consent was obtained from each participant.In the current study the slowing respiratory rate to 6 breaths/min increased HR (P<0.001) (not shown), decreased SBP (P<0.001), decreased DBP (P=0.003), increased LnLF (P<0001), increased TV (P<0.001), and decreased RR (not shown, Р<0.001). The stepwise multiple linear regression analysis revealed that at rest HR and RR were negatively associated with LnHF power (Table 1, P<0.001 for both), while at slow breathing stage the HR was negatively associated with both LnHF and LnLF power (Table 2, P<0.001 and P=0.004, respectively). If the HF power is widely used as an index of cardiovagal activity, interpretation of the LF power is more controversal. We proceeded from our hypothesis that LF HRV power is a measure of baroreflex‐mediated sympathoinhibition. At rest both SBP and DBP were positively associated with LnLF by LnHF interaction, while at slow breathing stage they were negatively associated only with LnLF power. The resting TV and MV were positively associated with LnLF power, also, the MV with LnHF by height and BMI interaction. But at slow breathing stage only the height and BMI were independent variables remaining in these models (Table 2, Models 4 and 5).Taken together, these findings indicate that the same autonomic variables control respiratory function in parallel with cardiovascular system.
Mutation in the 5'SL Region of Collagen I mRNA Attenuates Diastolic Dysfunction in Male, but not Female, Mice Following 28 Weeks of Feeding a High Fat High Sucrose Diet
Authors: Russell Jacob , Bender Shawn B. , Ramirez‐Perez Francisco , Woodford Makenzie , Ruff Margot , Brenner David , Yoshida Tadashi , DeMarco Vincent , Martinez‐Lemus Luis , Padilla Jaume , Chandrasekar Bysani 
Published: May 2022

IntroductionThe La ribonucleoprotein 6 (LARP6), an RNA binding protein, increases type I collagen expression by regulating mRNA stability and translation. This is mediated via its interaction with the 5’ stem loop (5’SL) structure in collagen I mRNA. Disrupting this interaction by mutating the 5’SL region has been shown to reduce fibrotic remodeling in liver. Therefore, we tested the hypothesis that this interaction plays a role in cardiac fibrosis and stiffening in diet induced obesity in 5’SL mutant and wild‐type littermate mice. In fact, obesity and hypertension promote remodeling and cardiac stiffening due to excess collagen deposition, fibrosis, and microvascular dysfunction, that ultimately lead to diastolic dysfunction.MethodsMale and female 5’SL mice and wild‐type (WT) littermates were fed a high fat high sucrose (HFHS) or control diet starting at 6 weeks of age. After 28 weeks of feeding, mice were assessed for cardiac function by echocardiography. Hearts were collected and a transverse section of the left and right ventricle was fixed for histology. RNA was isolated from the left ventricle and submitted for mRNA sequencing and qPCR.ResultsFollowing 28 weeks of a HFHS diet, male and female mice showed increased body weight, cardiac hypertrophy (heart weight normalized to tibia length), and diastolic dysfunction. However, the 5’SL mutation attenuated the impaired diastolic function in male, but not female mice. Surprisingly, cardiac fibrosis was not altered by the HFHS diet. RNA sequencing revealed that HFHS diet altered gene expression associated with glucose and ketone metabolism, as well as inflammation, in male WT littermates. However, the 5’SL mutation enriched gene networks associated with vascular development and angiogenesis.ConclusionsChronic HFHS feeding leads to increased body weight, hypertrophic cardiomyopathy, and diastolic dysfunction in both male and female mice. However, the 5’SL mutation blunts dysfunction only in male mice, demonstrating sex‐dependent cardioprotective effects.