19 research outputs found
Previously differentiated medial vascular smooth muscle cells contribute to neointima formation following vascular injury
Background
The origins of neointimal smooth muscle cells that arise following vascular injury remains controversial. Studies have suggested that these cells may arise from previously differentiated medial vascular smooth muscle cells, resident stem cells or blood born progenitors. In the current study we examined the contribution of the previously differentiated vascular smooth muscle cells to the neointima that forms following carotid artery ligation.
Methods
We utilized transgenic mice harboring a cre recombinase-dependent reporter gene (mTmG). These mice express membrane targeted tandem dimer Tomato (mTomato) prior to cre-mediated excision and membrane targeted EGFP (mEGFP) following excision. The mTmG mice were crossed with transgenic mice expressing either smooth muscle myosin heavy chain (Myh11) or smooth muscle Îą-actin (Acta2) driven tamoxifen regulated cre recombinase. Following treatment of adult mice with tamoxifen these mice express mEGFP exclusively in differentiated smooth muscle cells. Subsequently vascular injury was induced in the mice by carotid artery ligation and the contribution of mEGFP positive cells to the neointima determined.
Results
Analysis of the cellular composition of the neointima that forms following injury revealed that mEGFP positive cells derived from either Mhy11 or Acta2 tagged medial vascular smooth muscle cells contribute to the majority of neointima formation (79âÂąâ17% and 81âÂąâ12%, respectively).
Conclusion
These data demonstrate that the majority of the neointima that forms following carotid ligation is derived from previously differentiated medial vascular smooth muscle cells
Gastroparesis is associated with decreased FOXF1 and FOXF2 in humans, and loss of FOXF1 and FOXF2 results in gastroparesis in mice
Background and Aims
The transcription factors FOXF1 and FOXF2 have been implicated in the development of the gastrointestinal tract but their role in adults or in gastrointestinal diseases is poorly understood. We have recently shown that expression of serum response factor (SRF), a transcription factor whose activity is modulated by FOXF proteins, is decreased in the stomach muscularis of patients with gastroparesis. The aim of the current study was to determine whether FOXF expression is decreased in gastroparesis patients and whether loss of FOXF1 and/or FOXF2 from adult smooth muscle is sufficient to impair gastric emptying in mice.
Methods
Fullâthickness stomach biopsy samples were collected from control subjects and from patients with gastroparesis. mRNA was isolated from the muscularis externa, and FOXF mRNA expression levels were determined by quantitative reverse transcriptase (RT)âPCR. Foxf1 and Foxf2 were knocked out together and separately from smooth muscle cells in adult mice, and the subsequent effect on liquid gastric emptying and contractile protein expression was determined.
Key Results
Expression of FOXF1 and FOXF2 is decreased in smooth muscle tissue from gastroparesis patients. Knockout of Foxf1 and Foxf2 together, but not alone, from mouse smooth muscle resulted in delayed liquid gastric emptying. Foxf1/2 double knockout mice had decreased expression of smooth muscle contractile proteins, SRF, and myocardin in stomach muscularis.
Conclusions and Inferences
Our findings suggest that decreased expression of FOXF1 and FOXF2 may be contributing to the impaired gastric emptying seen in gastroparesis patients
Transcriptome profiling reveals significant changes in the gastric muscularis externa with obesity that partially overlap those that occur with idiopathic gastroparesis
BACKGROUND:
Gastric emptying is impaired in patients with gastroparesis whereas it is either unchanged or accelerated in obese individuals. The goal of the current study was to identify changes in gene expression in the stomach muscularis that may be contributing to altered gastric motility in idiopathic gastroparesis and obesity.
METHODS:
Quantitative real time RT-PCR and whole transcriptome sequencing were used to compare the transcriptomes of lean individuals, obese individuals and either lean or obese individuals with idiopathic gastroparesis.
RESULTS:
Obesity leads to an increase in mRNAs associated with muscle contractility whereas idiopathic gastroparesis leads to a decrease in mRNAs associated with PDGF BB signaling. Both obesity and idiopathic gastroparesis were also associated with similar alterations in pathways associated with inflammation.
CONCLUSIONS:
Our findings show that obesity and idiopathic gastroparesis result in overlapping but distinct changes in the gastric muscularis transcriptome. Increased expression of mRNAs encoding smooth muscle contractile proteins may be contributing to the increased gastric motility observed in obese subjects, whereas decreased PDGF BB signaling may be contributing to the impaired motility seen in subjects with idiopathic gastroparesis
Regulation of 130kDa smooth muscle myosin light chain kinase expression by an intronic CArG element
The mylk1 gene encodes a 220-kDa nonmuscle myosin light chain kinase (MLCK), a 130-kDa smooth muscle MLCK (smMLCK), as well as the non-catalytic product telokin. Together, these proteins play critical roles in regulating smooth muscle contractility. Changes in their expression are associated with many pathological conditions; thus, it is important to understand the mechanisms regulating expression of mylk1 gene transcripts. Previously, we reported a highly conserved CArG box, which binds serum response factor, in intron 15 of mylk1. Because this CArG element is near the promoter that drives transcription of the 130-kDa smMLCK, we examined its role in regulating expression of this transcript. Results show that deletion of the intronic CArG region from a β-galactosidase reporter gene abolished transgene expression in mice in vivo. Deletion of the CArG region from the endogenous mylk1 gene, specifically in smooth muscle cells, decreased expression of the 130-kDa smMLCK by 40% without affecting expression of the 220-kDa MLCK or telokin. This reduction in 130-kDa smMLCK expression resulted in decreased phosphorylation of myosin light chains, attenuated smooth muscle contractility, and a 24% decrease in small intestine length that was associated with a significant reduction of Ki67-positive smooth muscle cells. Overall, these data show that the CArG element in intron 15 of the mylk1 gene is necessary for maximal expression of the 130-kDa smMLCK and that the 130-kDa smMLCK isoform is specifically required to regulate smooth muscle contractility and small intestine smooth muscle cell proliferation
Lrp4 Mediates Bone Homeostasis and Mechanotransduction through Interaction with Sclerostin In Vivo
Wnt signaling plays a key role in regulating bone remodeling. In vitro studies suggest that sclerostin's inhibitory action on Lrp5 is facilitated by the membrane-associated receptor Lrp4. We generated an Lrp4 R1170W knockin mouse model (Lrp4KI), based on a published mutation in patients with high bone mass (HBM). Lrp4KI mice have an HBM phenotype (assessed radiographically), including increased bone strength and formation. Overexpression of a Sost transgene had osteopenic effects in Lrp4-WT but not Lrp4KI mice. Conversely, sclerostin inhibition had blunted osteoanabolic effects in Lrp4KI mice. In a disuse-induced bone wasting model, Lrp4KI mice exhibit significantly less bone loss than wild-type (WT) mice. In summary, mice harboring the Lrp4-R1170W missense mutation recapitulate the human HBM phenotype, are less sensitive to altered sclerostin levels, and are protected from disuse-induced bone loss. Lrp4 is an attractive target for pharmacological targeting aimed at increasing bone mass and preventing bone loss due to disuse
Idiopathic gastroparesis is associated with specific transcriptional changes in the gastric muscularis externa
BACKGROUND:
The molecular changes that occur in the stomach that are associated with idiopathic gastroparesis are poorly described. The aim of this study was to use quantitative analysis of mRNA expression to identify changes in mRNAs encoding proteins required for the normal motility functions of the stomach.
METHODS:
Full-thickness stomach biopsy samples were collected from non-diabetic control subjects who exhibited no symptoms of gastroparesis and from patients with idiopathic gastroparesis. mRNA was isolated from the muscularis externa and mRNA expression levels were determined by quantitative reverse transcriptase (RT)-PCR.
KEY RESULTS:
Smooth muscle tissue from idiopathic gastroparesis patients had decreased expression of mRNAs encoding several contractile proteins, such as MYH11 and MYLK1. Conversely, there was no significant change in mRNAs characteristic of interstitial cells of Cajal (ICCs) such as KIT or ANO1. There was also a significant decrease in mRNA-encoding platelet-derived growth factor receptor Îą (PDGFRÎą) and its ligand PDGFB and in Heme oxygenase 1 in idiopathic gastroparesis subjects. In contrast, there was a small increase in mRNA characteristic of neurons. Although there was not an overall change in KIT expression in gastroparesis patients, KIT expression showed a significant correlation with gastric emptying whereas changes in MYLK1, ANO1 and PDGFRÎą showed weak correlations to the fullness/satiety subscore of patient assessment of upper gastrointestinal disorder-symptom severity index scores.
CONCLUSIONS AND INFERENCES:
Our findings suggest that idiopathic gastroparesis is associated with altered smooth muscle cell contractile protein expression and loss of PDGFRÎą+ cells without a significant change in ICCs
Improving Bone Health by Optimizing the Anabolic Action of Wnt Inhibitor Multitargeting
Sclerostin antibody (romosozumab) was recently approved for clinical use in the United States to treat osteoporosis. We and others have explored Wntâbased combination therapy to disproportionately improve the anabolic effects of sclerostin inhibition, including cotreatment with sclerostin antibody (SclâmAb) and Dkk1 antibody (Dkk1âmAb). To determine the optimal ratio of SclâmAb and Dkk1âmAb for producing maximal anabolic action, the proportion of SclâmAb and Dkk1âmAb were systematically varied while holding the total antibody dose constant. A 3:1 mixture of SclâmAb to Dkk1âmAb produced two to three times as much cancellous bone mass as an equivalent dose of SclâmAb alone. Further, a 75% reduction in the dose of the 3:1 mixture was equally efficacious to a full dose of SclâmAb in the distal femur metaphysis. The SclâmAb/Dkk1âmAb combination approach was highly efficacious in the cancellous bone mass, but the cortical compartment was much more subtly affected. The osteoanabolic effects of Wnt pathway targeting can be made more efficient if multiple antagonists are simultaneously targeted. Š 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research
Expression of a DegradationâResistant βâCatenin Mutant in Osteocytes Protects the Skeleton From MechanodeprivationâInduced Bone Wasting
Mechanical stimulation is a key regulator of bone mass, maintenance, and turnover. Wnt signaling is a key regulator of mechanotransduction in bone, but the role of βâcateninâan intracellular signaling node in the canonical Wnt pathwayâin disuse mechanotransduction is not defined. Using the βâcatenin exon 3 flox (constitutively active [CA]) mouse model, in conjunction with a tamoxifenâinducible, osteocyteâselective Cre driver, we evaluated the effects of degradationâresistant βâcatenin on bone properties during disuse. We hypothesized that if βâcatenin plays an important role in Wntâmediated osteoprotection, then artificial stabilization of βâcatenin in osteocytes would protect the limbs from disuseâinduced bone wasting. Two disuse models were tested: tail suspension, which models fluid shift, and botulinumâtoxin (botox)âinduced muscle paralysis, which models loss of muscle force. Tail suspension was associated with a significant loss of tibial bone mass and density, reduced architectural properties, and decreased bone formation indices in uninduced (control) mice, as assessed by dualâenergy Xâray absorptiometry (DXA), microâcomputed tomography (ÂľCT), and histomorphometry. Activation of the βcatCA allele in tailâsuspended mice resulted in little to no change in those properties; ie, these mice were protected from bone loss. Similar protective effects were observed among botoxâtreated mice when the βcatCA was activated. RNAseq analysis of altered gene regulation in tailâsuspended mice yielded 35 genes, including Wnt11, Gli1, Nell1, Gdf5, and Pgf, which were significantly differentially regulated between tailâsuspended βâcatenin stabilized mice and tailâsuspended nonstabilized mice. Our findings indicate that selectively targeting/blocking of βâcatenin degradation in bone cells could have therapeutic implications in mechanically induced bone disease