bluuebunny/crossref_metadata_2025 huggingface dataset:
Protection of Glomeruli from Hyperhomocysteinemia‐Induced Injury in Acid Sphingomyelinase Gene Knockout Mice
Authors: Boini Krishna M , Zhang Chun , Xia Min , Li Pin‐Lan
Published: June 2021

Ceramide has been reported to initiate local oxidative injury in glomeruli under different pathological conditions. The present study was performed to explore the role of acid sphingomyelinase (ASM) in the development of hyperhomocysteinemia (hHcys)‐induced glomerular injury. Uninephrectomized ASM knockout (asm−/−) and wild type (asm+/+) mice were fed a folate free (FF) diet or normal chow for 8 weeks to produce hHcys. HPLC analysis revealed that plasma Hcys was significantly elevated in both asm−/− and asm+/+ mice. However, the plasma Hcys levels were significantly attenuated in asm−/−than inasm+/+ mice. FF diet significantly increased the renal ASM mRNA expression in asm+/+mice than in asm−/−mice. Morphological examinations showed that FF diet induced profound injury in glomeruli of asm+/+ mice which was markedly attenuated in asm−/− mice(the glomerular damage index (GDI) in asm+/+: 2.95± 0.09 vs. asm−/−: 1.51 ± 0.11). The decreased glomerular injury in asm−/− mice was accompanied by attenuated proteinuria associated with hHcy. Immunocytochemical analysis showed that FF diet decreased expression of podocin and nephrin, but increased desmin and ceramide levels in glomeruli of asm+/+ mice than in asm−/− mice. Electron spin resonance analysis demonstrated that FF diet significantly increased the NADPH‐dependent superoxide (O2·−) production in the kidneys of asm+/+ than in asm−/− mice. In conclusion, the observations reveal a pivotal role of ASM in the hHcy‐induced glomerular injury and ASM may be a therapeutic target for treatment or prevention of glomerulosclerosis associated with hHcys (supported by NIH grants HL‐091464, HL‐75316 and DK54927).
Renal Vascular Responses Are Attenuated in Smooth Muscle‐Specific Na+/Ca2+ Exchanger Knockout Mice during Acute Angiotensin II Infusions
Authors: Zhao Di , Blaustein Mordecai P , Navar L. Gabriel
Published: June 2021

Studies in smooth muscle‐specific Na+/Ca2+ exchanger knockout (NCX1sm−/−) mice reveal reduced arterial pressure and impaired myogenic responses compared to heterozygous littermates. In this study, we determined renal function in male anesthetized NCX1sm−/− mice (n=6) and global NCX1 heterozygous (NCX1+/−, n=8) littermates before and during acute Ang II infusions (5 ng/g Body Weight/min). Systolic blood pressure in awake mice was lower in NCX1sm−/− mice compared to NCX1+/− mice (119±4 versus 131±3 mm Hg, P<0.05). Acute Ang II infusions increased mean arterial pressure in NCX1sm−/− (98±8 to 123±8 mm Hg, P<0.001) and NCX1+/− mice (109±2 to 134±3 mm Hg, P<0.001). Acute Ang II infusions did not change renal plasma flow (RPF; Δ0.06±0.09 ml/min, P>0.05) or glomerular filtration rate (GFR; Δ0.003±0.04 ml/min, P>0.05) in NCX1sm−/−mice, whereas acute Ang II infusions significantly decreased RPF (Δ−0.2±0.09 ml/min, P<0.05) and GFR (Δ−0.08±0.03 ml/min, P<0.05) in NCX1+/− mice. The urine flow responses to acute Ang II infusions were significantly greater in NCX1sm−/−mice as compared with NCX1+/− mice (Δ1.88±0.83 versus Δ‐0.33±0.42 μl/min, P<0.05). Urine flow and urinary sodium excretion were higher in NCX1sm−/− mice as compared with NCX1+/− mice during acute Ang II infusions. These data suggest that renal vascular responses to Ang II are attenuated in NCX1sm−/− mice as compared with NCX1+/− mice and that NCX1 contributes to the renal vasoconstriction response to acute Ang II infusions.

Supported by NIH/NHLBI.

Disclosure: no conflict of interest
Renal Function of Rats with Isoproternol‐induced Cardiac Hypertrophy
Authors: Thieme Karina , Oliveira‐Souza Maria
Published: June 2021

Aim

Avaliate the effect of Isoproterenol‐induced cardiac hypertrophy on renal function and proteic expression in rats.

Methods

Male Wistar rats were submitted to subcutaneous injection of isoproterenol (ISO) (0.3 mg.kg(‐1).day(‐1) sc) or vehicle for 7 days. Renal plasmatic flow (RPF) and glomerular filtration rate (GFR) were determined by para aminohypurate and inulin clearance. The excretion of ions was also analyzed. Cardiac hypertrophy and organs congestion were evaluated by respective organs weights. The proteic expression of many renal transporters and Angiotensin II receptors will be analysed by Western Blot technique.

Results and Discussion

Chronic β‐adrenergic stimulation does not affect hemodynamic parameters, despite cardiac hypertrophy. Our results showed that ISO‐treatment does not affect RPF (control: 4,19 ± 0,45; n = 12 versus ISO: 4,17 ± 0,36; n = 11), which is consistent with a normal cardiac output found in this model. However, there was a significant decrease of GFR (control: 2,24 ± 0,22; n = 12 versus ISO: 1,49 ± 0,13; n = 11 * p< 0.01), which could be related to inflammatory process, leading to alteration in ultratiltration coefficient (Kf). Urinary Na+ and K+ were significantly reduced and enhanced, respectively. These changes in excretion of ions could be related to alterations in expression of renal transporters and are under investigation.

Research Support: Fapesp
Role of the SNARE protein SNAP‐23 on cAMP‐stimulated Renin release from mouse Juxtaglomerular (JG) cells
Authors: Mendez Mariela , Garvin Jeffrey L , Carretero Oscar A
Published: June 2021

Exocytosis of renin containing granules is likely involved in renin release from juxtaglomerular (JG) cells; a process stimulated by cAMP. In non‐neuronal endocrine cells granule exocytosis is mediated by SNAP‐23, a protein of the soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) family. We hypothesized that cAMP‐stimulated renin release from JG cells is in part mediated by SNAP‐23. We measured: 1) the expression of SNAP‐23 by Western blot and immunofluorescence confocal microscopy; and 2) the effect of adenoviral delivery of a dominant‐negative SNAP‐23 and botulinum toxin E on cAMP‐stimulated renin release in mouse JG cell primary cultures. cAMP was increased with Forkolin plus IBMX (F/I). SNAP‐23 was detected by Western blot in JG cell lysates and co‐localized with renin granules in JG cells. In control JG cells transduced with a scramble sequence, increasing cAMP stimulated renin release from 1.3±0.3 to 5.3±1.2 % of renin content. In cells transduced with dominant‐negative SNAP‐23, F/I increased renin from 1.0±0.1 to 3±0.6 % of renin content, a 50% blockade (p<0.02). Botulinum toxin E, which cleaves and inactivates SNAP‐23, reduced cAMP‐stimulated renin release from 5.0±0.7 to 2.45±0.5 % of renin content, a 50% blockade (p< 0.05). We concluded that the SNARE protein SNAP‐23 partially mediates cAMP‐stimulated renin release. These data show for the first time that renin release is mediated by SNAREs.
Reduced 22Na+ Uptake and Dysregulation of NHERF2/Akt/NOS Pathway in Rat Renal Proximal Tubule Cells Stably Overexpressing p22phox
Authors: Luo Zaiming , Panico Carolina , Artigiano Francesca , Gutweiler Alex , Jose Pedro A. , Palm Fredrik , Welch William J. , Wilcox Christopher S.
Published: June 2021

We showed that superoxide (O2−) reduced renal proximal tubule (PT) reabsorption (Hypertension. 2009 Oct.), but, the molecular mechanisms are unknown. We tested the hypothesis that O2− impairs Na+‐H+ exchanger 3 (NHE3) activity. A stably p22phox transfected rat PT cell line (s‐p22phox), compared to empty vector transfected wild type cells (Wt), had increased p22phox mRNA and protein by 4.1‐ and 1.59‐fold, and O2− by 2.2‐ fold but reduced 22Na+uptake by 36% (all P<0.05). Immunoblot assay of s‐p22phox cells showed no change in NHE3 but increased expression of inhibitory Na+–H+ exchanger regulatory factor 2 (NHERF2), neuronal and inducible nitric oxide synthase (nNOS & iNOS) by 183%, 293% and 372%, respectively (all P<0.05). Since activated Akt regulates NOS and NHERF2, we compared the Akt in both cell types. Total Akt (t‐Akt) protein abundance was similar but the phosphorylated Akt at ser473 (p‐Aktser473) was markedly increased leading to a 207% higher p‐Akt/t‐Akt ratio in s‐p22phox cells. Knockdown of NHEFR2 by specific siRNA in s‐p22phox cells increased significantly (25±4.9%; P<0.05) 22Na+uptake. In conclusion, overexpression of p22phox in PT cells inhibits NHE3 transport activity and upregulates NHERF2, nNOS and iNOS, and phosphorylated Akt. This dysregulation of NHERF2/Akt/NOS signaling pathways by O2− impairs PT function. This provides a novel insight into the molecular mechanisms underlying the effects of oxidative stress on ion and fluid homeostasis in the kidney.
Amelioration of glomerulosclerosis by NMDA receptor blockade in hyperhomocysteinemic rats
Authors: Zhang Chun , Xia Min , Krishna Krishna M , Hu Jun‐Jun , Laperle Laura A , Li Pin‐Lan
Published: June 2021

We have reported that hyperhomocyteinemia (hHcys) serves as a pathogenic factor to induce glomerulosclerosis. However, it remains unknown whether homocysteine (Hcys) exerts its actions through specific receptors. The present study tested the hypothesis that NMDA receptor (NMDAR) activation is involved in the development of hHcys‐induced glomerulosclerosis. Uninephrectomized Sprague‐Dawley rats were fed a folate‐free diet for 8 weeks to produce hHcys and an antagonist for NMDAR, MK‐801, was administrated. Rats treated with a folate‐free diet exhibited significantly increased plasma Hcys level (from 5.041±0.478 to 16.187±3.172), upregulation of glomerular NMDAR‐1 and NMDAR‐2A, enhanced NADPH oxidase (Nox)‐dependent O2.‐ production (2.8 folds vs. control), and remarkable glomerulosclerosis. Administration of MK‐801 significantly reduced these renal functional and pathological changes induced by hHcys. Correspondingly, extracellular matrix (ECM) deposition in the glomeruli of hHcys rats was ameliorated by the treatment with MK‐801, which was accompanied by changes in the activity of ECM proteolytic enzymes such as tissue inhibitor of metalloproteinase‐1 and matrix metalloproteinase‐1 and −9, towards a normal level. These results suggest that NMDAR is essential in Hcys‐induced Nox activation and consequent development of glomerulosclerosis (supported by NIH grants DK54927 and HL075316).
MitoQ protects against cold ischemic injury in renal cells and rat kidneys
Authors: Mitchell Tanecia , Rotaru Dumitru , Saba Hamida , Murphy Michael , MacMillan‐Crow Lee Ann
Published: June 2021

Cold preservation is a beneficial and necessary procedure used to preserve deceased donor kidneys prior to transplantation. However, extended preservation periods result in injury that can lead to poor graft function following transplantation. Thus, this study's purpose was to evaluate the early effects of cold storage (CS) alone and to determine if MitoQ, a mitochondrial targeted antioxidant, could offer protection during this stage. We hypothesize that CS increases oxidant production and mitochondrial dysfunction, and that adding MitoQ to cold preservation solutions will attenuate injury. Renal cells and rat kidneys were exposed to CS (UW‐Viaspan solution; 4°C) with or without MitoQ (1μM; 100μM respectively) and monitored for oxidants (O2·−, NO, ONOO−), mitochondrial function (respiratory complexes, mitochondrial membrane potential), and cell viability. Our cell model results suggest that CS first causes mitochondrial membrane depolarization, followed by oxidant production, then mitochondrial dysfunction, and modest cell death. MitoQ reduced oxidant production and protected against mitochondrial complex inactivation using both cellular and isolated rat kidney models. Excitingly, we have identified a potential therapeutic reagent that could be added to existing preservation solutions to reduce CS damage and possibly improve renal function following transplantation.
Increases in Cardiac Output Recruit Intrapulmonary Shunt Pathways in the Rat
Authors: Bates Melissa , Pegelow David , Fulmer Brendan , Drezdon Alyssa , Farrell Emily , Eldridge Marlowe
Published: June 2021

Using an intact, spontaneously breathing, instrumented rat model, we investigated the effects of progressive embolization and pulmonary blood flow on intrapulmonary shunt pathway (IPAVs) recruitment. We hypothesized that progressive embolization and increased regional flow would recruit IPAVs in a dose‐dependent manner. We injected six boluses of 5×105 15μm spheres (3×106 total) in 0.4 or 0.8 mL aliquots via the inferior vena cava and harvested the kidney to assess transpulmonary passage of spheres. Spheres bypassed the lungs in 1/3 rats and 3/3 rats injected with 0.4 and 0.8 mL, respectively. The shunt fraction increased in a dose‐dependent manner with progressive embolization. Interestingly, right ventricular systolic pressure (RVSP) rose in the rats receiving 0.4mL injections (28±4 to 36±15 mmHg), but not in animals receiving 0.8 mL (24±3 mmHg vs 24±1mmHg). The transiently higher cardiac output associated with the larger volume injections may recruit and distend the pulmonary vasculature, including IPAVs, resulting in lower RVSP and greater sphere passage. These data suggest that increased pulmonary blood flow is important for IPAV recruitment. Further studies are needed to better define the mechanical and mediator‐based mechanisms regulating IPAVs and their importance in determining pulmonary vasculature pressure.

This research was supported by a grant from the NIH (5R01HL086897‐02).
Functional interaction of ClCa with RyR and CaL in pulmonary arteries from chronic hypoxic sheep
Authors: Vemulakonda Srilakshmi , Papamatheakis Demosthenes G. , Forrest Abigail , Leblanc Normand , Angermann Jeff E. , Longo Lawrence D. , Wilson Sean M.
Published: June 2021

Agonists such as serotonin (5‐HT) mediate pulmonary artery (PA) contraction, in large part, through membrane depolarization, activation of L‐type Ca2+ channels (CaL) and subsequent cytosolic Ca2+ increases. The sequence leading from 5‐HT receptor stimulation through to contraction remains controversial. Experimental evidence suggests that calcium activated chloride channels (ClCa) are pivotal to the depolarization response and that ryanodine receptors (RyRs) are important to the activity of ClCa. These associations led us to test the hypothesis that ClCa‐mediated contractility is dependent on coordination with RyR as well as CaL. These hypotheses were tested by performing wire‐myography on isolated PA from adult chronic hypoxic sheep, where hypoxia was induced by housing sheep at 3,801 m for 100+ days. 5‐HT elicited dose‐dependent contractions were attenuated by inhibitors for ClCa (100 μM niflumic acid, NFA), CaL (10 μM nifedipine, NIF), and RyR (10 μM dantrolene, DAN) separately and combined. NIF alone or the combinations of NIF+NFA or NFA+DAN reduced 5‐HT elicited contractility to similar extents. NFA or DAN alone blocked contractility less effectively. These results support the proposal that 5‐HT receptor activation promotes PA contractility in chronic hypoxic sheep through the cooperative activity of RyR, ClCa and CaL. NIH P01HD031226, R01HD003807 (LDL), and R01 HL075477, NCRR P20 RR15581 (NL)
Perinatal Nicotine Exposure‐Induced Upper Airway and Lung Parenchymal Structural and Functional Changes are Blocked by Concomitant PPARγ Agonist Administration
Authors: Rehan Virender K. , Liu Jie , Sakurai Reiko , Torday John S.
Published: June 2021

Recently, we have demonstrated the down‐regulation of the homeostatic alveolar mesenchymal PPARγ signaling and the up‐regulation of Wnt signaling in offspring lung following perinatal exposure to nicotine (NC). However, the molecular and functional alterations in upper airways in this setting are not known. Our objectives were to determine the effects of a PPARγ agonist on perinatal nicotine exposure‐induced alterations in 1) mesenchymal markers of tracheal constriction, and 2) acetylcholine‐induced tracheal constriction responses. Pair‐fed pregnant Sprague Dawley rat dams received diluent, NC (1 mg/kg, sc), and NC (1 mg/kg, sc) + a PPARγ agonist, Rosiglitazone (RGZ) (3 mg/kg, i.p), once daily from e6 until the pups were 21‐days old, when the pups were sacrificed and tracheas collected for mRNA/protein expression and tracheal tension studies. Compared to control, with perinatal NC exposure, mRNA and protein expression of α‐smooth muscle actin, calponin, collagen 1 and 3 were significantly increased. Similarly, compared to controls, tracheal constriction responses were also significantly increased. Concomitant treatment with RGZ blocked the NC‐induced alterations in both tracheal constriction marker expression and constriction responses. Alterations in upper airway function in a rodent model of perinatal NC exposure were blocked by concomitant administration of RGZ. We speculate that perinatal smoke exposure‐induced asthma in offspring can be effectively prevented by PPARγ agonists. Disclosure: Supported by grants from the NIH (HL75405, HD51857, HD058948) and the TRDRP (14RT‐0073, 15IT‐0250, 17RT‐0170).
Neuregulin improves neuromuscular transmission in diaphragm muscle of young rats
Authors: Ermilov Leonid G , Mantilla Carlos B , Sieck Dylan C , Zhan Wen‐Zhi , Sieck Gary C
Published: June 2021

Neuregulin (NRG) is important for expression of acetylcholine (ACh) receptors during development of the neuromuscular junction (NMJ). NRG treatment significantly increases ACh‐induced currents in hippocampal neurons, but it is not known if NRG exerts a similar effect at NMJs. Diaphragm muscle (DIAm)‐phrenic nerve preparations from 7‐ and 14‐day old rats were used to determine the effects of NRG (heregulin β177–244) on neuromuscular transmission and DIAm contractile properties. Neuromuscular transmission failure (NMTF) was assessed by the difference in force elicited by repetitive phrenic nerve stimulation (0.2 ms pulses in 40 Hz, 330 ms trains) and superimposed, intermittent direct DIAm stimulation (1 ms pulses in 40 Hz, 330 ms trains applied every 15 seconds). NMTF was higher in 7‐day old compared to 14‐day old rats (76% vs. 65%; p<0.05). Exogenous NRG treatment significantly improved neuromuscular transmission in 7‐day old rats (NMTF: 52%; p<0.05), but not in 14‐day old rats (65%). Isometric DIAm contractile properties were not affected by NRG treatment in either age group. These results indicate that NRG has important synaptic effects during early postnatal development of the DIAm. Whether NRG effects on neuromuscular transmission result from acute changes in AChR expression or alternative mechanisms remains to be explored.

Supported by NIH grant AR51173 and a Career Development Award from Mayo Clinic.
Neurotrophins improve neuromuscular transmission in rats with myasthenia gravis
Authors: Mantilla Carlos B , Ermilov Leonid G , Zhan Wen‐Zhi
Published: June 2021

Myasthenia gravis is a disease of the neuromuscular junction (NMJ) associated with autoimmune destruction of the motor end‐plate. Inhibitors of acetylcholine (ACh) esterase are commonly used to mitigate symptoms by increasing ACh availability at the NMJ. Brain‐derived neurotrophic factor (BDNF) improves neuromuscular transmission in the rat diaphragm muscle (DIAm) by maintaining ACh release with repeated activation. We hypothesized that BDNF will mitigate the deficit of neuromuscular transmission in a passive transfer model of myasthenia gravis. Female Lewis rats were injected with vehicle, McAb1 (that does not induce myasthenia), and a myasthenogenic McAb3 antibody. Neuromuscular transmission failure (NMTF) was determined in DIAm‐phrenic nerve preparations by comparing the isometric force evoked by phrenic nerve stimulation and that evoked by superimposed direct muscle stimulation. After 2 min of repetitive stimulation, neuromuscular transmission was significantly impaired in myasthenic animals (NMTF: 80%) compared to vehicle and McAb1 animals (NMTF: ~60%). Exogenous BDNF treatment significantly improved neuromuscular transmission in all groups and NMTF in McAb3‐treated rats was no longer different from control or McAb1‐treated animals. We conclude that BDNF improves neuromuscular transmission in adult myasthenic rats.

Supported by a Career Development Award from Mayo Clinic.
Chronic nicotinic exposure decreases the synaptic strength of nAChR‐mediated glutamatergic input onto neonatal hypoglossal motoneurons
Authors: Pilarski Jason Q. , Wakefield Hilary E. , Fregosi Ralph F.
Published: June 2021

Although nicotinic acetylcholine receptors (nAChRs) are widespread in the CNS their role in the control of breathing is still unclear. This study aims to test the hypothesis that alterations in nAChRs, evoked by chronic nAChR excitation, impairs respiratory‐related neurotransmission onto hypoglossal motoneurons (HMN) that control the tongue. To test our hypothesis we used rhythmic medullary slice preparations and simultaneously recorded from both single respiratory modulated HMN and whole hypoglossal nerve rootlets on day 1–4 in rat pups chronically exposed to nicotine or saline in utero and during the first week of life. Results show that synaptic strength, predominately glutamatergic, was decreased but without changes in the frequency of respiratory rhythm determined by the respiratory central pattern generator (CPG). The average number of spontaneous excitatory postsynaptic potentials (EPSPs), measured during the “expiratory” period, was decreased 42% (P < 0.05), but their magnitude was unchanged. Furthermore, EPSPs could be abolished by bath application of AMPAergic antagonist 6‐cyano‐7‐nitroquinoxaline‐2, 3‐dione (CNQX). These results show that the chronic in utero exposure to nicotine decreases excitatory neurotransmission at HMN synapses and suggests that this change results from frequency of nAChR‐mediated presynpatic release of glutamate. Funded by NIH #1R03HD061613‐01.
Mathematical Modeling of Calcium Signaling in Porcine Coronary Arteries
Authors: Francis Michael , Solodushko Viktoriya , Taylor Mark S.
Published: June 2021

The vascular endothelium continuously regulates smooth muscle tone through several calcium‐dependent mechanisms. In porcine coronary artery endothelial cells, calcium is released basally from intracellular stores by the inositol 1,4,5‐trisphosphate receptor (IP3R). This release forms a widespread, slowly decaying event with a peak near the nucleus. Our objective was to investigate the relationship between the spatial and temporal dynamics of calcium signaling and the intracellular distribution of IP3R. To determine the distribution of IP3R, we performed immunofluorescence targeting the distinct IP3R isoforms. We measured the time course of intracellular calcium events, and the spatial relationship between the event magnitude and IP3R distribution. We found that intracellular calcium events had an average time of 21 sec and amplitude of 2.5 times baseline. Event peaks were located within 2μm of dense IP3R clusters of isoforms I and II, adjacent to the nucleus. To study the effect of the intracellular distribution of IP3R, we developed a deterministic model of IP3R‐dependent calcium release using Matlab software. Simulation of a clustered IP3R distribution had a substantial impact on the spread and duration of calcium events compared to a continuous IP3R distribution, and reproduced wave‐like behavior with longstanding calcium increase at IP3R clusters. Supported by NIH HL085887 and AHA 0435051N.
A polymer chip‐based technology for the investigation of small resistance arteries
Authors: Voigtlaender‐Bolz Julia , Yasotharan Sanjesh , Vagaon Andrei , Lochovsky Conrad , Guenther Axel , Bolz Steffen‐Sebastian
Published: June 2021

Growing consensus links hypertension, a primary cardiovascular risk factor, to elevated peripheral resistance. Advanced research on peripheral resistance arteries (RA) is therefore essential to improve our knowledge regarding hypertension and existing treatment strategies. Current experimental approaches (i.e., pressure myography) are time intensive, not high throughput ready and require highly skilled personnel. To overcome these limitations, we have designed and fabricated a microfluidic device that allows RAs to be reversibly loaded, fixed, and perfused on a polymer chip (AoC). The current study aims to validate the AoC in relation to the classical cannulation approach.

Dose response curves for phenylephrine (PE) and acetylcholine (ACh) were virtually identical for mesenteric RAs tested on the AoC and those studied on a conventional pressure myograph. Arteries kept in culture on the AoC for 24h prior to functional testing showed fully maintained responses to PE and ACh. On‐chip vessel loading with Fura‐2 revealed irregular Ca2+ oscillations that were synchronized following PE treatment.

This study presents a fast, easy to manage, low cost, scalable and high throughput‐ready technology that will drastically facilitate microvascular research. Based on its innovative features, the AoC has great potential for application in drug development and on‐site clinical use (personalized medicine).
Protein Tyrosine Nitration and Glutathionylation Impairs Pro‐Survival Mechanisms of Akt in CryABmut Cardiomyopathy in Mice
Authors: Soorappan Rajasekaran Namakkal , Varadharaj Saradhadevi , Zhang Huali , Zweier Jay L , Benjamin Ivor J
Published: June 2021

Background

Serine‐threonine Akt affords powerful cellular protection but the role of this pro‐survival pathway in hR120GCryAB‐TG cardiomyopathy remains poorly defined. We hypothesized that chronic oxidative modifications and sequestration of Akt could impair the pro‐survival mechanisms in the hR120GCryAB‐TG leading to heart failure.

Methods

To gain further insights, we have determined biochemical markers of oxidative modifications.

Results

Western blot and immunohistochemical analyses indicated increased protein kinase‐B (PKB/Akt) is sequestered into the protein aggregates containing hR120GCryAB in myopathic hearts compared with the non‐transgenic (NTG) controls. Increased S‐glutathionylation and tyrosine nitrosylation were present in the 3 and 6 months old TG mice, suggesting potential role in inactivation of pro‐survival mechanisms of Akt. Surprisingly, we found increased Akt kinase (substrate: GSK3‐α/β) activity in vitro in the hR120GCryAB‐TG hearts compared with the NTG, suggesting chronic Akt activation is coupled with cardiac hypertrophy and progression of cardiaomyopathy.

Conclusions

Taken together, these findings implicate chronic activation and sequestration of Akt in the pathogenesis of hR120GCryAB‐induced cardiotoxicity.
Bone marrow cell therapy modulates remodeling of the myocardium remnant to experimental infarction in the rat: effects on the contractility and calcium handling proteins
Authors: dos Santos Leonardo , Gonçalves Giovana A. , Santos Alexandra A. , Davel Ana P. , Krieger Jose E. , Rossoni Luciana V. , Tucci Paulo J.
Published: June 2021

We evaluated the effects of bone marrow‐derived mononuclear cells (MNC) on the left ventricular (LV) function, and functional and molecular remodeling of tissue remnant to myocardial infarct (MI) in the rat. Saline (MI group) or 6×106 MNC (MI+MNC) were injected in the infarct border zone of Lewis inbred rats 48h after coronary occlusion and compared to a sham group (SO). After 6 weeks, infarct size of MI+MNC (38±2%) turned smaller than MI (44±2%), and LV dilatation and dysfunction were partially attenuated. Interestingly, developed force of isolated LV muscle samples was impaired in MI (2.6±0.4g/mm2) but fully preserved in MI+MNC (5.6±0.7) when compared to SO (5.9±0.5), in spite of sizable infarcts. Contractile responses to calcium and beta‐agonist were better in muscles from MI+MNC, as well as the post‐rest potentiation of force. Concordantly, decreases in sarcoplasmic Ca2+ATPase (46%), total (47%) and Ser16‐phosphorylated phospholamban (65%) were prevented by MNC (5, 10 and 9% respectively). While no changes were noted on α1‐Na+K+ATPase, decrease in α2 isoform due to MI was not influenced by MNC. We added evidences that MNC injection restricts post‐MI cardiac dysfunction. Also, our results indicated that this therapy preserved mechanics of myocardium remnant to infarct associated to prevention on specific calcium handling protein alterations.

Supported by FAPESP and CNPq.
Adenosine attenuates the lipopolysaccharide (LPS) induced endothelial barrier dysfunction in murine model of acute lung injury
Authors: Gonzales Joyce N , Gorshkov Boris A , Umapathy NS , Black Stephen M , Verin Alexander D
Published: June 2021

Rationale

Acute lung injury (ALI) affects approximately 200,000 people in the United States every year. A basic cause is pulmonary endothelial cell (EC) barrier disruption and the ensuing inflammation and increased permeability of the EC. Our previous studies on the human pulmonary arterial endothelial cells have shown that adenosine (AD), an endogenous purine nucleoside, attenuates the EC barrier. The goal of our experiments was to evaluate AD attenuation on the EC barrier in LPS treated mice.

Results

Our preliminary data indicate that mice challenged with LPS demonstrated an inflammatory response typical for ALI compared with normal saline (NS) treated controls. Microscopic examination of bronchoalveolar (BAL) showed fewer neutrophils in control and LPS/AD treated mice compared to LPS challenged mice. Protein and EBD extravasation analysis showed a decrease in the LPS/AD treated mice compared to LPS alone.

Conclusions

AD attenuated the LPS‐induced permeability increase as evidenced by decreased leakage of protein and in the BAL and EBD extravasation into the lung. In addition, AD also decreased lung inflammation measured by BAL neutrophil cell counts. Further studies into the AD‐mediated signaling mechanisms in protecting the EC barrier may help elucidate the molecular pathways involved in EC barrier regulation. This work was supported by NIHLBI grant HL 083327 (ADV) and MCG PSRP grant (NSU).
Vascular dysfunction in adult mice generated by assisted reproductive technologies
Authors: Rexhaj Emrush , Scherrer Urs , Sartori Claudio
Published: June 2021

Environmental influences acting early in life may predispose to premature cardiovascular disease. Assisted reproductive technologies (ART) involve the manipulation of early embryos at a time when they may be particularly vulnerable to external disturbances. We hypothesized ART predisposes to vascular dysfunction later in life.

We therefore assessed vascular function in vitro (mesenteric artery) and in vivo (blood pressure, carotid artery) in young adult (10 wks) ART and control mice.

In ART mice endothelium‐dependent mesenteric‐artery vasodilation in vitro was markedly impaired (P<.001 vs. control). This endothelial dysfunction in vitro was associated with arterial hypertension in vivo (108±4 vs. 103±4 mmHg, X±SE, P=.02, ART vs. ctrl). Most interestingly, male ART offspring transmitted their vascular dysfunction to the next generation, suggesting an epigenetic mechanism. To test for this possibility, we examined the effects of the histone deacetylase inhibitor Butyrate (2 mg/kg/day, i.p. for 2 wks) to male offspring of ART. Butyrate normalized vascular function in these animals (P<.001 vs. ctrls) and prevented the transmission of this defect to the next generation (P<0.001 butyrate vs. vehicle). We demonstrate for the first time that ART induces premature systemic vascular dysfunction in mice by an epigenetic mechanism. We speculate that ART may induce a similar problem in humans.

Grant Funding Source: Swiss National Science Foundation
Re‐Expression of locomotion and reflex changes after various types of spinal lesions
Authors: Rossignol Serge
Published: June 2021

Locomotion of the hindlimbs is a complex motor act involving several levels of control (supraspinal, sensory, neurochemical) of a spinal circuitry called the Central Pattern Generator (CPG) which essentially generates the basic locomotor pattern. The importance of this CPG is quite evident from experiments showing that cats with a complete spinal section at T13 can gradually recover spontaneous hindlimb locomotion on a treadmill with appropriate locomotor training for a few weeks. The key role played by the CPG, and some reflex changes, in the recovery of locomotion after a complete spinal section is thus inescapable. Is the spinal CPG also involved in the re‐expression of locomotion after partial spinal lesions? A dual lesion paradigm was devised to study this question: hemisection at T10‐T11, locomotor training and then complete section at T13. Our main finding was that, within 24 hours (i.e. the first testing session) following the complete spinal section, treadmill‐trained cats walked remarkably well with the hindlimbs on the treadmill. Thus, the present experiments show that there is also a major functional contribution from the CPG in the recovery of locomotion after partial spinal cord lesions. This highlights the importance of fostering such potential spinal neuroplasticity in rehabilitation strategies by reinforcing both voluntary aspects of locomotion and activity in endogenous spinal circuits.
Bone Marrow‐Derived Cells Stimulate Healing by Modulating Early Inflammatory Processes
Authors: Schatteman Gina C , Jiao Chunhua , Leymaster Nicholas , Timpe Josh , Dunnwald Martine
Published: June 2021

Improvements in healing and vascular growth from acute exogenous bone marrow‐derived cell (BMDC) therapy are apparent by a few days after injury, suggesting that BMDCs modulate early inflammatory events. That MCP‐1 is essential for BMDC mediated healing supports this hypothesis. To examine BMDC impact on inflammatory processes and subsequent growth factor production, BMDCs from healthy (wt), obese diabetic (db) and monocyte chemoattractant protein‐1 (MCP‐1‐) knockout mice were injected into ischemic mouse hindlimbs. (BMDCs from C57Bl/6 but not db or MCP‐1‐ mice stimulate MCP‐1 production and promote limb healing.) Fewer immunolabeled monocytes (day 3) and neutrophils (day 1) were seen in ischemic limbs treated with BMDCs from db and MCP‐1‐ mice than those that received cells from wt mice. However, levels of six pro‐angiogenic growth factors measured by ELISA at days 1, 3, and 5 did not correlate with the extent of monocyte/neutrophil infiltration or healing. We observed a complex temporally regulated pattern of growth factor expression in ischemic muscle that differed depending on cell treatment, but at all time points for all factors differences were at most 2‐fold. Thus, BMDCs may stimulate healing by an immunomodulatory process that only modestly impacts pro‐angiogenic growth factor expression and that may not require their upregulation to induce vascular growth and healing.

Support: NIH DK55965
Disambiguating Muscular Forces Effecting Hyoid Movement in Pharyngeal Phase of Deglutition
Authors: Pearson William G. , Zumwalt Ann
Published: June 2021

Introduction

Superior and anterior hyoid movements are important clinical events in the pharyngeal phase of swallowing. Fluoroscopy charts the movement of the bone but cannot assess soft tissue. Electromyography specifies muscle activation but not the structural effect of depolarization. This cross‐sectional study uses a cadaver model to generate the continuous data needed to establish an anatomical foundation for these movements.

Methods

Measurements to determine physiological cross‐sectional areas (PCSA) of swallowing muscles were taken from 7 formalin‐fixed cadavers. Cartesian attachment coordinates and PCSAs were used to calculate i and j unit force vectors where i and j represent anterior and superior movements, respectively. Three functional muscle groups were established for analysis of variance (ANOVA): mylohyoid and stylohyoid (MH‐SH), digastrics (DG), and geniohyoid (GH).

Results

ANOVA with Tukey HSD showed a statistical difference in i unit force vectors (GH > DG [p < .01], GH > MH‐SH [p < .01], DG > MH‐SH [p < .05]) and j unit force vectors (MH‐SH > DG [p < .01], MH‐SH > GH [p < .01]).

Discussion

The GH is primarily responsible for the anterior excursion of the hyoid during swallowing while the MH‐SH elevates the hyoid. This is the first in a series of investigations to elucidate the anatomical substrate of pharyngeal deglutition to enable clinicians to better understand and treat dysphagia.

Grant Funding Source: n/a
Migration and differentiation of epicardial cells stimulated by TGFβ1, TGFβ2, or BMP‐2 do not require the Type III Transforming Growth Factor β receptor
Authors: Austin Anita , Compton Leigh A , Love Joseph D , Sanchez Nora A , Brown Christopher B , Barnett Joey V
Published: June 2021

Transforming Growth Factor β (TGFβ) Receptor III (TGFβR3) binds all three TGFβ ligands, inhibin, and Bone Morphogenic Protein‐2 (BMP‐2). Since TGFβ causes epicardial cell differentiation into smooth muscle in vitro we asked if Tgfbr3 null epicardial cells differentiate in response to TGFβ or BMP‐2. Wiltype and Tgfbr3−/− immortalized mouse epicardial cells (ED 11.5) were cultured in the presence or absence of ligands for 72 hours and scored by immunohistochemistry, QT‐PCR, or a reportor assay for Smooth Muscle 22 alpha (SM22α). Wildtype and Tgfbr3−/− cells lose epithelial character and undergo smooth muscle differentiation in response to 250 pM TGFβ1 or TGFβ2. Induction of SM22α in wildtype cells occurred with an EC50 of 12.5 pM. BMP‐2 (5 nM) induced the loss of Zonula Occludens‐1 (ZO‐1) but failed to induce the smooth muscle markers SM22α, smooth muscle α‐actin (SMαA) or calponin. Inhibition of activin receptor‐like kinase (ALK) 5 with 2.5 μM SB431542 blocked the effects of TGFβ1 and TGFβ2, but not BMP‐2. Pre‐ and co‐incubation of cells with 5 nM BMP‐2 did not block the ability of 250 pM TGFβ1 or TGFβ2 to induce smooth muscle markers. These data demonstrate that BMP2 induces epicardial cell motility without smooth muscle differentiation and that TGFβR3 is not required for epicardial cell responsiveness to TGFβ or BMP‐2.

Funding: HL085708, NIGMS 5K12GM068543

Grant Funding Source: NIH HL
Nerve and Vascular Regeneration in Bone Transport Osteogenesis
Authors: Malavia Veera , Kontogiorgos E , Elsalanty ME , Dechow PC , Opperman LA
Published: June 2021

Objective

To evaluate blood vessel and nerve formation in mandibular regenerate bone reconstructed by distraction osteogenesis (DO).

Methods

In 10 dogs, a 30–40mm mandibular defect was created. A bone transport reconstruction device was used to create regenerate across the gap, using standard DO criteria, followed by 6 week of consolidation. Three implants were placed on the regenerate and control sides. One group (n=5) was left to heal for 12 weeks and the other (n=5) for 18 weeks. After sacrifice, regenerate and control bone was prepared for histology and n=2–3 slides/group were analyzed. One examiner blinded to the experiment counted numbers of blood vessels and nerves.

Results

Regenerate bone had significantly more blood vessels than control bone. Increased vascularity of the regenerate appeared to be due to the formation of new small blood vessels. Healing time had no effect on the number of blood vessels in the regenerate. Nerve number increased from 12 to 18 weeks for both groups. In the control group the number of small nerves increased; the regenerate group showed an increase in small and large nerves.

Conclusion

The increased vascularity of the regenerate relates to new bone regeneration. Increased innervation of regenerate appears due to nerve generation in new bone, while in controls nerve damage due to implant placement might increase small nerve sprouting.

Grant Funding Source: Supported by grants DE017259 and DE15437 from NIDCR
Mechanism and regulation of RNA Polymerase II transcription
Authors: Hahn Steven
Published: June 2021

Recruitment of the RNA polymerase II (Pol II) transcription machinery at promoters involves the coordinated action of transcription activators and coactivators that facilitate positioning of Pol II and the general transcription factors at a promoter. This preinitiation complex (PIC) is poised to undergo a conformational change into an active conformation (open complex) with the melted template DNA strand in the enzyme active site. To understand the recruitment and transcription initiation steps, we have solved the structure of an activator‐coactivator complex and used biochemical probes to model the architecture of the PIC containing the general factor TFIIF.

The yeast activator Gcn4 contains tandem acidic activation domains that interact with the Mediator coactivator subunit Gal11/Med15. The central Gcn4 activation domain adopts a helical structure when bound to Gal11 with three hydrophobic residues contacting a small hydrophobic surface of Gal11 with micromolar affinity. Unlike other activator‐target structures, there are no observed electrostatic interactions to help determine Gcn4 orientation. This simple and versatile interface can explain how acidic activators interact with multiple targets.

The general transcription factor TFIIF functions at several steps in transcription initiation including PIC formation and start site selection. We find that in the PIC, two structured TFIIF domains bind Pol II at separate locations far from the active site with the TFIIF dimerization domain on the Pol II lobe and the winged helix domain of the TFIIF small subunit Tfg2 above the Pol II protrusion. Anchoring of these two structured TFIIF domains at separate sites locates an essential and unstructured region of Tfg2 near the Pol II cleft where it is in a position to interact with flexible regions of Pol II and the general factor TFIIB to promote initiation and start site selection.
Mechanisms of Hormonal Regulation of the Sodium Chloride Cotransporter
Authors: Hoover Robert
Published: June 2021

The thiazide‐sensitive sodium chloride cotransporter (NCC) is one of the key determinants of salt balance and thus systemic blood pressure. Aldosterone and Angiotensin II appear to regulate NCC. However, in contrast to other important effectors of salt balance we know little about how hormonal signals alter activity of this cotransporter. Data demonstrates the role of the ERK1/2 MAPK (Extra‐cellular signal‐Regulated Kinases 1 and 2 Mitogen‐Activated Protein Kinases) pathway in the ubiquitination and endocytosis of NCC. With‐No‐Lysine Kinase 1 and 4 (WNK1 and WNK4), Ste20‐related proline alanine‐rich kinase (SPAK) and Oxidative Stress Response‐1 (OSR1) are also important regulators of NCC, and recent publications suggests that they may be linked to hormonal effects on NCC. However, the examination of links between hormones and the intracellular pathways that mediate the effects of hormones on NCC is only now beginning. Our recent data provides evidence that NCC is subject to hormonal regulation that proceeds through modulation of kinases.
RNA folding during assembly of the 30S ribosome
Authors: Woodson Sarah A , Adilakshmi Tadepalli , Ramaswamy Priya , Soper Sarah F. C. , Bellur Deepti
Published: June 2021

The biogenesis of new ribosomal subunits is a highly regulated process essential for cell growth. During assembly of the ribosomal subunits, ribosomal proteins join the complex in a specific hierarchy as the rRNA folds. We used hydroxyl radical footprinting to visualize changes in the structure of the 16S rRNA during assembly in real time. The results show that each domain of the 30S ribosome assembles concurrently in vitro, and that many tertiary RNA interactions and RNA‐protein interactions are established within the first 100 milliseconds. A single protein can stabilize the folded structure of the entire 16S 5' domain. However, the assembly includes both native and non‐native conformations of the 16S rRNA. Protein S16 preferentially stabilizes a native‐like assembly intermediate and promotes a conformational switch at helix 3 that stabilizes the decoding center of the 30S ribosome. In vivo footprinting experiments are beginning to reveal the conformations of immature subunits that accumulate when one or more 30S assembly factors are missing.
The Role of Microbial Communities in Health and Disease
Authors: Fraser‐Liggett Claire M
Published: June 2021

The human species is dependent for its survival upon the activities of billions of microorganisms that inhabit multiple environmental niches within and on the human body. Our current view of microbial‐host interactions is extremely limited by the fact that the overwhelming majority of microbial species (>99%) resists cultivation in the laboratory. Their identification and characterization requires the use of molecular approaches. The impact of this vast number of uncharacterized microbes on human health and disease is potentially significant and remains to be elucidated. The identification and characterization of these microbial communities will undoubtedly establish links between these microorganisms and disease, their roles in development of the immune system, and their overall impact on human evolution. The Human Microbiome Project (HMP), which is a collection of projects around the world, is just being launched with the ultimate goals being to better understand our microbiota and to ultimately manipulate it so as to optimize its beneficial effects on each individual. In this presentation, I will focus on a number of the conceptual and experimental challenges associated with the HMP, with particular emphasis on the human gastrointestinal tract, since this environment is home to the largest number of our microbial partners. (This work was funded in part by NIH grants R01 DE014868 and P01 DK078669).
Isolation and purification of Human Milk Oligosaccharides by two‐dimensional chromatography for in vitro and in vivo studies
Authors: Jantscher‐Krenn Evelyn , Nissan Caroline , Bode Lars
Published: June 2021

Human Milk Oligosaccharides (HMO) are a structurally divers group of complex glycans that are highly abundant in human milk but not in infant formula. The structural diversity of HMO, however, makes it difficult to study the molecular mechanisms underlying their potential benefits for the breast‐fed infant. More than 150 different HMO have been described ranging in size from low molecular weight trisaccharides to high molecular weight oligosaccharides with more than 30 monosaccharide monomers and ranging from being neutral to carrying several negative charges. Not all HMO may trigger the same effects. Here, we describe the isolation and purification of HMO subfractions and even individual HMO by two‐dimensional chromatography. In the first dimension we used anion‐exchange chromatography (QAE) to separate pooled HMO in a neutral and 5 negatively charged fractions containing 0 to 5 sialic acids per HMO molecule, respectively. In the second dimension we used automated size exclusion chromatography to separate HMO based on their molecular weight. Structural composition of the generated HMO fractions was analyzed by HPLC and mass spectrometry. Our approach generated structurally defined HMO subfractions and even individual HMO in quantities sufficient for in vitro and in vivo experiments aimed to elucidate HMO structure‐function relationships. (Supported by NIH K99/R00DK078668 to LB)
Human milk oligosaccharides prevent Necrotizing Enterocolitis in neonatal rats
Authors: Bode Lars , Goth Kerstin , Guner Yigit , Nissan Caroline , Zherebtsov Monica , Jantscher‐Krenn Evelyn , Grishin Anatoly , Ford Henri
Published: June 2021

Necrotizing Enterocolitis (NEC) is the most common and often fatal intestinal disorder in preterm infants. Breast‐fed infants are at much lower risk to develop NEC than formula‐fed infants. We hypothesize that Human Milk Oligosaccharides (HMO), complex glycans highly abundant in breast milk but not in formula, contribute to the protective effects of breast‐feeding. In an established NEC model neonatal Sprague‐Dawley rats were either left with the dam to serve as breast‐fed control or orally gavaged with formula without and with HMO. All pups were exposed to hypoxia thrice a day, sacrificed on day 4, and their intestines analyzed for macroscopic and microscopic signs of NEC. Histology sections of the terminal ileum were scored blindly from 0 (healthy) to 4 (complete destruction). The average pathology score in breast‐fed pups was 0.15+/−0.34. It was significantly increased to 1.98+/−1.11 in pups receiving formula without HMO (P<0.001). Addition of HMO reduced the pathology score to 0.44+/−0.30, which was significantly lower than in the group receiving formula without HMO (P<0.001) but not different from the breast‐fed control. Galactooligosaccharides (GOS), currently added to most infant formula to mimic HMO, had no effect. We are now identifying individual HMO responsible for the beneficial effects and aim to understand the underlying molecular mechanisms. (Supported by NIH K99/R00DK078668 to LB)