scholarly journals In vivo magnetofection: a novel approach for targeted topical delivery of nucleic acids for rectoanal motility disorders

2018 ◽  
Vol 314 (1) ◽  
pp. G109-G118 ◽  
Author(s):  
Jagmohan Singh ◽  
Ipsita Mohanty ◽  
Satish Rattan
Keyword(s):  
Smooth Muscle ◽  
Gene Delivery ◽  
Nucleic Acids ◽  
Internal Anal Sphincter ◽  
Fecal Pellet ◽  
Muscle Layer ◽  
Motility Disorders ◽  
Circular Smooth Muscle ◽  
Novel Approach

In these studies, we developed a novel approach of in vivo magnetofection for localized delivery of nucleic acids such as micro-RNA-139-5p (miR-139-5p; which is known to target Rho kinase2) to the circular smooth muscle layer of the internal anal sphincter (IAS). The IAS tone is known to play a major role in the rectoanal continence via activation of RhoA-associated kinase (RhoA/ROCK2). These studies established an optimized protocol for efficient gene delivery using an assembly of equal volumes of in vivo PolyMag and miR139-5p or anti-miR-139-5p (100 nM each) injected in the circular smooth muscle layer in the pinpointed areas of the rat perianal region and then incubated for 20 min under magnetic field. Magnetofection efficiency was confirmed and analyzed by confocal microscopy of FITC-tagged siRNA. Using physiological and biochemical approaches, we investigated the effects of miR-139-5p and anti-miR-139-5p on basal intraluminal IAS pressure (IASP), fecal pellet count, IAS tone, agonist-induced contraction, contraction-relaxation kinetics, and RhoA/ROCK2 signaling. Present studies demonstrate that magnetofection-mediated miR-139-5p delivery significantly decreased RhoA/ROCK2, p-MYPT1, and p-MLC20 signaling, leading to decreases in the basal IASP and IAS tone and in rates of contraction and relaxation associated with increase in fecal pellet output. Interestingly, anti-miR-139-5p transfection had opposite effects on these parameters. Collectively, these data demonstrate that magnetofection is a promising novel method of in vivo gene delivery and of nucleotides to the internal anal sphincter for the site-directed and targeted therapy for rectoanal motility disorders. NEW & NOTEWORTHY These studies for the first time demonstrate the success of topical in vivo magnetofection (MF) of nucleic acids using perianal injections. To demonstrate its effectiveness, we used FITC-tagged siRNA via immunofluorescence microcopy and functional and biochemical evidence using miR-139-5p (which is known to target ROCK2). In conclusion, MF allows safe, convenient, efficient, and targeted delivery of oligonucleotides such as siRNAs and microRNAs. These studies have direct therapeutic implications in rectoanal motility disorders especially associated with IAS.

Mechanical properties of isolated smooth muscle from human rectum and internal anal sphincter

1993 ◽  
Vol 265 (4) ◽  
pp. G792-G798 ◽  
Author(s):  
E. B. Glavind ◽  
A. Forman ◽  
G. Madsen ◽  
D. Svane ◽  
K. E. Andersson ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Anal Sphincter ◽  
Internal Anal Sphincter ◽  
Muscle Layer ◽  
Isometric Tension ◽  
Passive Tension ◽  
Active Tension ◽  
Active Length ◽  
Circular Smooth Muscle ◽  
Resting Tension

The passive and active length-tension relations of the circular smooth muscle layer of the human distal rectum and the proximal and distal internal anal sphincter were investigated. Muscle strips were prepared and mounted in organ baths for recording of isometric tension. Resting lengths (LR) were measured, and the preparations were elongated stepwise. At each length, the corresponding values for passive tension, spontaneous active resting tension, and the submaximal active tension were recorded. Elongations of 200-380% of LR were possible before a sharp increase in passive tension occurred. None of the mean tension values measured at length for maximal active tension (LO) differed significantly among the three muscle types. All strips developed active resting tension. This tension was myogenic and contributed 10 +/- 3, 23 +/- 6, and 27 +/- 6% to the total active performance of rectal and proximal and distal sphincter preparations, respectively. Collagen constituted approximately 50% of smooth muscle biopsies, with highest contents in distal internal anal sphincter. This study provides an acceptable method for assessing the optimal experimental length by stretching the strips in an inactive state to 200% of LR, followed by individual adjustment of the passive tension to 5 mN/mm2 measured at 200% of LR

Exosome as a Natural Gene Delivery Vector for Cancer Treatment

2020 ◽  
Vol 20 (11) ◽  
pp. 821-830
Author(s):  
Prasad Pofali ◽  
Adrita Mondal ◽  
Vaishali Londhe
Keyword(s):  
Gene Therapy ◽  
Gene Delivery ◽  
Nucleic Acids ◽  
Cancer Treatment ◽  
Intestinal Barrier ◽  
Gene Carrier ◽  
Gene Delivery Vector ◽  
Delivery Vector

Background: Current gene therapy vectors such as viral, non-viral, and bacterial vectors, which are used for cancer treatment, but there are certain safety concerns and stability issues of these conventional vectors. Exosomes are the vesicles of size 40-100 nm secreted from multivesicular bodies into the extracellular environment by most of the cell types in-vivo and in-vitro. As a natural nanocarrier, exosomes are immunologically inert, biocompatible, and can cross biological barriers like the blood-brain barrier, intestinal barrier, and placental barrier. Objective: This review focusses on the role of exosome as a carrier to efficiently deliver a gene for cancer treatment and diagnosis. The methods for loading of nucleic acids onto the exosomes, advantages of exosomes as a smart intercellular shuttle for gene delivery and therapeutic applications as a gene delivery vector for siRNA, miRNA and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and also the limitations of exosomes as a gene carrier are all reviewed in this article. Methods: Mostly, electroporation and chemical transfection are used to prepare gene loaded exosomes. Results: Exosome-mediated delivery is highly promising and advantageous in comparison to the current delivery methods for systemic gene therapy. Targeted exosomes, loaded with therapeutic nucleic acids, can efficiently promote the reduction of tumor proliferation without any adverse effects. Conclusion: In the near future, exosomes can become an efficient gene carrier for delivery and a biomarker for the diagnosis and treatment of cancer.

scholarly journals Aging-associated changes in microRNA expression profile of internal anal sphincter smooth muscle: Role of microRNA-133a

2016 ◽  
Vol 311 (5) ◽  
pp. G964-G973 ◽  
Author(s):  
Jagmohan Singh ◽  
Ettickan Boopathi ◽  
Sankar Addya ◽  
Benjamin Phillips ◽  
Isidore Rigoutsos ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Anal Sphincter ◽  
Internal Anal Sphincter ◽  
Smooth Muscles ◽  
Smooth Muscle Contractility ◽  
Network Analyses ◽  
Rhoa Signaling

A comprehensive genomic and proteomic, computational, and physiological approach was employed to examine the (previously unexplored) role of microRNAs (miRNAs) as regulators of internal anal sphincter (IAS) smooth muscle contractile phenotype and basal tone. miRNA profiling, genome-wide expression, validation, and network analyses were employed to assess changes in mRNA and miRNA expression in IAS smooth muscles from young vs. aging rats. Multiple miRNAs, including rno-miR-1, rno-miR-340-5p, rno-miR-185, rno-miR-199a-3p, rno-miR-200c, rno-miR-200b, rno-miR-31, rno-miR-133a, and rno-miR-206, were found to be upregulated in aging IAS. qPCR confirmed the upregulated expression of these miRNAs and downregulation of multiple, predicted targets ( Eln, Col3a1, Col1a1, Zeb2, Myocd, Srf, Smad1, Smad2, Rhoa/Rock2, Fn1, Tagln v2, Klf4, and Acta2) involved in regulation of smooth muscle contractility. Subsequent studies demonstrated an aging-associated increase in the expression of miR-133a, corresponding decreases in RhoA, ROCK2, MYOCD, SRF, and SM22α protein expression, RhoA-signaling, and a decrease in basal and agonist [U-46619 (thromboxane A2analog)]-induced increase in the IAS tone. Moreover, in vitro transfection of miR-133a caused a dose-dependent increase of IAS tone in strips, which was reversed by anti-miR-133a. Last, in vivo perianal injection of anti-miR-133a reversed the loss of IAS tone associated with age. This work establishes the important regulatory effect of miRNA-133a on basal and agonist-stimulated IAS tone. Moreover, reversal of age-associated loss of tone via anti-miR delivery strongly implicates miR dysregulation as a causal factor in the aging-associated decrease in IAS tone and suggests that miR-133a is a feasible therapeutic target in aging-associated rectoanal incontinence.

Abstract P501: Editing of Myosin Phosphatase Gene as a Novel Approach for Vasodilator Sensitization and Lowering of Blood Pressure

Hypertension ◽  
2017 ◽  
Vol 70 (suppl_1) ◽  
Author(s):  
Mariam Meddeb ◽  
Jeanine Ursitti ◽  
John Reho ◽  
Steven A Fisher
Keyword(s):  
Blood Pressure ◽  
Smooth Muscle ◽  
Leucine Zipper ◽  
Splice Variants ◽  
Species Conservation ◽  
Mesenteric Arteries ◽  
Hek293 Cells ◽  
Myosin Phosphatase ◽  
Novel Approach

Myosin Phosphatase (MP) is the primary effector of vascular smooth muscle (VSM) relaxation and a key end target of signaling pathways that regulate vessel tone. Regulated splicing of alternative Exon24 (E24) of Myosin Phosphatase Regulatory/ Targeting subunit (MYPT1) sets vasodilator sensitivity. Skipping E24 codes for a Mypt1 isoform that contains a C-terminal leucine zipper (LZ) motif required for cGK1α binding and NO/cGMP activation of MP resulting in vasodilation. Inclusion of 31 nt E24 shifts the reading frame coding for a Mypt1 isoform with a distinct C-terminus (LZ-) that is unresponsive to NO/cGMP. We are using two editing approaches to test the function of Mypt1 E24 splice variants in the control of BP in vivo. First, LoxP sites were inserted in introns flanking E24, crossed with smMHCCre ER , and treated with Tamoxifen to achieve smooth muscle-specific cKO of E24 (SMcKO E24), thereby converting Mypt1 to the LZ+ isoform. E24 cKO mice had mean BP that was 15 + 3 mmHg lower than control (n=3-5; p<0.05). Mesenteric arteries from these mice were significantly more sensitive to DEA/NO mediated relaxation (EC 50 : 2.1+0.5 nM vs 18.2+5.6 μM; n=5-6, p<0.05). We now are developing CRISPR/CAS9 editing of Mypt1 for translation into humans with hypertension. Guide(g)RNAs targeting E24 were designed using Benchling.com and selected for further study based on predicted efficacy, specificity (>10%,>60%) and cross-species conservation. Plasmids were generated by sub-cloning of oligonucleotides into the parent pX601 plasmid for the purpose of co-expression of gRNA and saCas9. These plasmids were transfected into HEK293 cells singly and in combinations and Mypt1 gene editing assayed by PCR, Surveyor nuclease assays and sequencing of genomic DNA. Single gRNAs yielded deletions of 1-3 nt. Combinations yielded deletions of 104-334 nt that removed >80% of E24 with an efficiency of editing that varied from 10% (gRNAs 6+9 and 5+9) to 40% (gRNAs 6+11 and 5+11). We have now generated AAVgE24 and are testing their efficiency of editing of VSM in vivo. These studies support that AAV mediated CRISPR/Cas9 editing of Mypt1 E24 could be a novel strategy for vasodilator sensitization and effective lowering of blood pressure in humans.

scholarly journals Ultrastructural and immunohistochemical localization of plasma membrane Ca2+-ATPase 4 in Ca2+-transporting epithelia

2015 ◽  
Vol 309 (7) ◽  
pp. F604-F616 ◽  
Author(s):  
R. Todd Alexander ◽  
Megan R. Beggs ◽  
Reza Zamani ◽  
Niels Marcussen ◽  
Sebastian Frische ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Transient Receptor Potential ◽  
Basolateral Membrane ◽  
Receptor Potential ◽  
Human Kidney ◽  
Muscle Layer ◽  
Transient Receptor Potential Vanilloid ◽  
Distal Nephron

Plasma membrane Ca2+-ATPases (PMCAs) participate in epithelial Ca2+ transport and intracellular Ca2+ signaling. The Pmca4 isoform is enriched in distal nephron isolates and decreased in mice lacking the epithelial transient receptor potential vanilloid 5 Ca2+ channel. We therefore hypothesized that Pmca4 plays a significant role in transcellular Ca2+ flux and investigated the localization and regulation of Pmca4 in Ca2+-transporting epithelia. Using antibodies directed specifically against Pmca4, we found it expressed only in the smooth muscle layer of mouse and human intestines, whereas pan-specific Pmca antibodies detected Pmca1 in lateral membranes of enterocytes. In the kidney, Pmca4 showed broad localization to the distal nephron. In the mouse, expression was most abundant in segments coexpressing the epithelial ransient receptor potential vanilloid 5 Ca2+ channel. Significant, albeit lower, expression was also evident in the region encompassing the cortical thick ascending limbs, macula densa, and early distal tubules as well as smooth muscle layers surrounding renal vessels. In the human kidney, a similar pattern of distribution was observed, with the highest PMCA4 expression in Na+-Cl− cotransporter-positive tubules. Electron microscopy demonstrated Pmca4 localization in distal nephron cells at both the basolateral membrane and intracellular perinuclear compartments but not submembranous vesicles, suggesting rapid trafficking to the plasma membrane is unlikely to occur in vivo. Pmca4 expression was not altered by perturbations in Ca2+ balance, pointing to a housekeeping function of the pump in Ca2+-transporting epithelia. In conclusion, Pmca4 shows a divergent expression pattern in Ca2+-transporting epithelia, inferring diverse roles for this isoform not limited to transepithelial Ca2+ transport.

Hyperoxia-induced airway hyperresponsiveness and remodeling in immature rats

1992 ◽  
Vol 262 (3) ◽  
pp. L263-L269 ◽  
Author(s):  
M. B. Hershenson ◽  
S. Aghili ◽  
N. Punjabi ◽  
C. Hernandez ◽  
D. W. Ray ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Airway Hyperresponsiveness ◽  
Muscle Layer ◽  
Airway Responsiveness ◽  
Wall Thickening ◽  
Small Airways ◽  
Smooth Muscle Layer ◽  
Hyperoxic Exposure ◽  
Immature Rats

We exposed 21-day-old rats to either normoxia or hyperoxia (greater than 95% O2) for 8 days and assessed in vivo airway responsiveness to aerosolized and intravenous methacholine (MCh) and airway architecture. Airway responsiveness was determined using a plethysmographic method. Hyperoxia increased airway cholinergic responsiveness, as reflected in a decreased mean ED200 (concentration of MCh required to increase respiratory system resistance by 100%) for both aerosolized MCh [air exposed, 5.94 +/- 2.50 vs. O2 exposed, 0.29 +/- 3.34 (SD) mg/ml, P = 0.0013, unpaired t test] and intravenous MCh (air, 1.40 x 10(-8) vs. O2, 2.45 x 10(-10) mol/kg, P = 0.0002). Airway morphometry was studied in a separate cohort of animals. After fixation by distension with Formalin at 25 cmH2O pressure, each airway cross section was photographed, and airway circumference, epithelial area, and smooth muscle layer area were determined by means of contour tracing using a digitizing pad and microcomputer. For the small airways (circumference less than 1,000 microns), hyperoxia increased both mean epithelial thickness (air, 4.88 +/- 0.53; O2, 8.64 +/- 0.90 microns) and mean smooth muscle layer thickness (air, 2.69 +/- 0.11; O2, 4.79 +/- 0.56 microns; P less than 0.0001 for each). O2 had similar effects on the larger (1,000-3,000 microns) central airways (P less than 0.0001 for both layers). We conclude that chronic hyperoxic exposure induces both airway hyperresponsiveness and airway wall thickening in immature rats.

Origin of Motion in the Human Ureter: Mechanics, Energetics and Kinetics of the Myosin Molecular Motors

2012 ◽  
Vol 79 (2) ◽  
pp. 123-129 ◽  
Author(s):  
Romina Vargiu ◽  
Anna Perinu ◽  
Antonello De Lisa ◽  
Frank Tintrup ◽  
Francesco Manca ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Molecular Motors ◽  
Circular Muscle ◽  
Muscle Layer ◽  
Shortening Velocity ◽  
Smooth Muscle Layer ◽  
Circular Smooth Muscle ◽  
Longitudinal Smooth Muscle ◽  
Human Ureter

Background Ureteral peristalsis is the result of coordinated mechanical motor performance of longitudinal and circular smooth muscle layer of the ureter wall. The main aim of this study was to characterize in smooth muscle of proximal segments of human ureter, the mechanical properties at level of muscle tissue and at level of myosin molecular motors. Methods Ureteral samples were collected from 15 patients, who underwent nephrectomy for renal cancer. Smooth muscle strips longitudinally and circularly oriented from proximal segments of human ureter were used for the in vitro experiments. Mechanical indices including the maximum unloaded shortening velocity (Vmax), and the maximum isometric tension (P0) normalized per cross-sectional area, were determined in vitro determined in electrically evoked contractions of longitudinal and circular smooth muscle strips. Myosin cross-bridge (CB) number per mm2 (Ψ) the elementary force per single CB (Ψ) and kinetic parameters were calculated in muscle strips, using Huxley's equations adapted to nonsarcomeric muscles. Results Longitudinal smooth muscle strips exhibited a significantly (p<0.05) faster Vmax (63%) and a higher P0 (40%), if compared to circular strips. Moreover, longitudinal muscle strips showed a significantly higher unitary force (Ψ) per CB. However, no significant differences were observed in CB number, the attachment (f1) and the detachment (g2) rate constants between longitudinal and circular muscle strips. Conclusions The main result obtained in the present work documents that the mechanical, energetic and unitary forces per CB of longitudinal layer of proximal ureter are better compared to the circular one; these preliminary findings suggested, unlike intestinal smooth muscle, a major role of longitudinal smooth muscle layer in the ureter peristalsis.

scholarly journals  Expression of corticotropin releasing factor (CRF) in the enteric nervous system of the jejunum of sheep

2011 ◽  
Vol 56 (No. 11) ◽  
pp. 551-560 ◽  
Author(s):  
A. Czujkowska ◽  
MB Arciszewski
Keyword(s):  
Nervous System ◽  
Smooth Muscle ◽  
Muscle Layer ◽  
Corticotropin Releasing Factor ◽  
Enteric Neurons ◽  
Nerve Fibres ◽  
Myenteric Neurons ◽  
Smooth Muscle Layer ◽  
Circular Smooth Muscle ◽  
A Minor

&nbsp;Corticotropin releasing factor (CRF), a 41-amino acid neuropeptide widely distributed in the mammalian central nervous system, has been shown to influence several gastrointestinal functions. Recent studies show that CRF released locally from enteric nerves may also underlie alterations in gut function. In this study, immunohistochemisty was applied to demonstrate the presence of CRF in the jejunum of sheep. Using double immunohistochemical staining the co-localization of CRF with vasoactive intestinal peptide (VIP), galanin, tyrosine hydroxylase (TH), neuropeptide&nbsp;Y (NPY) and substance P (SP) was evaluated. The presence of CRF was detected in myenteric neurons (3.6 &plusmn; 0.9%) as well as in submucous neurons (10.5 &plusmn; 1.2%). In the ovine jejunum different numbers of CRF-expressing nerve fibres were detected in myenteric ganglia, submucous ganglia, circular smooth muscle layer, lamina muscularis mucosae and between mucosal glands. None of the CRF-positive enteric neurons and CRF-positive nerve fibres exhibited the presence of TH. CRF-immunoreactive (IR) myenteric neurons widely co-expressed VIP and/or NPY. A minor population of CRF-IR myenteric neurons additionally co-stored SP. Galanin was not present in CRF-IR myenteric neurons. The presence of VIP was observed in the vast majority of CRF-positive submucous neurons. Moderate numbers of CRF-IR sumbucous neurons co-expressing galanin or NPY were also found. The presence of SP in CRF-positive submucous neurons was noted only incidentally. In the circular smooth muscle layer CRF-IR/VIP-IR, CRF-IR/NPY-IR as well as CRF-IR/SP-IR nerve fibres were present. In the mucosal layer of the ovine jejunum CRF-IR nerve fibres co-stored additionally VIP, galanin, NPY or SP. This present study provides for the first time evidence that CRF present in different subclasses of enteric neurons may influence certain activities of the ovine jejunum. Co-localization studies indicate that VIP, galanin, SP and NPY functionally co-operate with CRF in the jejunum of the sheep. &nbsp;

Biochemical and Cytochemical Characterization of Extracellular Proteoglycans in the Inner Circular Smooth Muscle Layer of Dog Small Intestine

IUBMB Life ◽  
2002 ◽  
Vol 54 (1) ◽  
pp. 19-25 ◽  
Author(s):  
Olga Maria Szymanski de Toledo ◽  
M&#x000F4;nica Val&#x000E9;ria Marquezini ◽  
Kaio Bin Jia ◽  
M&#x000F4;nica de Campos Pinheiro ◽  
Oswaldo Alves Mora
Keyword(s):  
Smooth Muscle ◽  
Small Intestine ◽  
Muscle Layer ◽  
Smooth Muscle Layer ◽  
Circular Smooth Muscle

scholarly journals KARAKTERISTIK MORFOLOGI DAN DISTRIBUSI KARBOHIDRAT NETRAL PADA UTERUS KELELAWAR BUAH (Pteropus vampyrus) ASAL PULAU TIMOR

2019 ◽  
Vol 7 (1) ◽  
pp. 80-84
Author(s):  
Yulfia Selan ◽  
Filphin A. Amalo ◽  
Inggrid Trinidad Maha ◽  
Antin Y. N. Widi ◽  
Cynthia D. Gaina ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Muscle Layer ◽  
Elastic Fibre ◽  
Secretory Cells ◽  
Epithelial Layer ◽  
Fruit Bats ◽  
Circular Smooth Muscle ◽  
Fruit Bat ◽  
Pteropus Vampyrus ◽  
Visceral Layer

Timorese fruit bat(Pteropusvampyrus)is the only fly mammalian with its unique behavior which hanging upside down inspite of its pregnancy. This research is aimed to reveal the morphology of the Timorese fruit bats and the distribution of neutral carbohydrate within this organ. Three uterus samples derived from three different Timorese fruit bats were used in the research.Both macroscopical and microscopical examinations using H&E and PAS methods were applied. Macroscopically, Timorese fruit bats showedsoft reddish white duplex uterus. Meanwhile microscopically, endometrium consisted of epithelial layer and lamina propria and was the place where simple tubular glands located. The epithelial layer comprised of simple cylindric secretory cells and ciliated cells. Neutral carbohydrate distribution was seen within this epithelial layer. Myometrium was a thick circular smooth muscle layer which consisted of smooth muscle separated by collagen and elastic fibre. Perimetrium was a visceral layer and consisted of mesothelial cells.

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