Dermal fibroblast-to-myofibroblast transition sustained by αvß3 integrin-ILK-Snail1/Slug signaling is a common feature for hypermobile Ehlers-Danlos syndrome and hypermobility spectrum disorders

May combine two claims.

CCN2/CTGF is present in the culture media of hEDS and HSD cells at levels similar to control and cEDS cells, but is found in higher amounts in hEDS and HSD cell extracts.

“CCN2/CTGF was present in the culture media of hEDS and HSD cells at levels comparable to those observed in control and cEDS cells, whereas in the CE it was found at higher amounts (Fig. 2C).”

May combine two claims.

Control and cEDS cells strongly express and organize CCN1/CYR61 on the plasma membrane and secrete it into the media.

“Control and cEDS cells abundantly expressed and organized on the plasma membrane CCN1/ CYR61, which was also secreted in the media (Fig. 2C).”

May combine two claims.

In hEDS and HSD cells, CCN1/CYR61 is not detectable in cell extracts and is present at low levels in their media.

“Conversely, in hEDS and HSD cells CCN1/CYR61 was undetectable in CE and present at low levels in their media (Fig. 2C).”

May combine two claims.

hEDS and HSD dermal fibroblasts arrange α-SMA into cytoskeletal stress fibres, while control, cEDS, and vEDS cells display disorganized α-SMA in the cytoplasm and perinuclear area.

“hEDS and HSD cells organized α-SMA in cytoskeletal stress fibres, whereas control, cEDS and vEDS cells showed, at different levels, disorganized α-SMA in the cytoplasm and perinuclear region.”

May combine two claims.

Control fibroblasts cultured with conditioned media from hEDS and HSD cells assemble α-SMA stress fibres in 40% to 85% of cells, whereas conditioned media from control and cEDS cells reduce α-SMA protein.

“In the presence of control and cEDS fibroblasts' CM, the cells showed a reduction of the α-SMA protein (Fig. 2A), whereas in the presence of CM recovered from hEDS and HSD cell strains from 40% to 85% of cells organized α-SMA stress fibres (Fig. 2A).”

May combine two claims.

Across different hEDS and HSD cell strains, the proportion of cells assembling an α-SMA cytoskeleton is roughly 60% to 90%, with no difference between hEDS and HSD or across in vitro passages.

“The percentage of cells organizing the α-SMA cytoskeleton ranged from about 60% to 90% in the different hEDS and HSD cell strains, without any difference between the two cell types and in vitro passage (not shown).”

May combine two claims.

hEDS and HSD fibroblasts express and organize OB-cadherin/cadherin-11 and αvβ3 integrin into the plasma membrane.

“hEDS and HSD fibroblasts express and organize into the plasma membrane other markers of the fibroblast-to-myofibroblast transition, i.e., OB-cadherin/cadherin-11 and αvβ3 integrin.”

May combine two claims.

hEDS/HSD fibroblast conditioned medium enhances migratory capability of control cells, resembling the hEDS/HSD myofibroblast-like phenotype.

“Furthermore, the CM of hEDS and HSD fibroblasts enhances the migratory capability of control cells, resembling the hEDS and HSD myofibroblast-like phenotype.”

May combine two claims.

Both hEDS and HSD skin fibroblasts display in vitro the same disorganized ECM, myofibroblast-like phenotype with α-SMA microfilaments, enhanced OB-cadherin/cadherin-11 and MMP-9, and αvβ3-mediated signaling involving ILK and Snail1/Slug.

“Indeed, both hEDS and HSD skin fibroblasts displayed in vitro the same disorganized ECM, a myofibroblast-like phenotype with the formation of α-SMA microfilaments, enhanced expression of OB-cadherin/cadherin-11 and MMP-9 together with an αvβ3-mediated signaling transduction pathway involving ILK and Snail1/Slug.”

May combine two claims.

Dermal fibroblasts from hEDS and HSD patients show a persistent myofibroblast-like phenotype with α-SMA stress fibres, which is absent in control, cEDS and vEDS fibroblasts.

“Overall, these data highlight that hEDS and HSD cells display a range of phenotypic features that are typical of myofibroblasts.”

• Also stated in: § 4. Discussion · plain

  “Skin fibroblasts isolated from hEDS and HSD patients show a persistent in vitro phenotype characterized by synthesis and organization of α-SMA in cytoskeletal stress fibres that is observed neither in cEDS and vEDS nor in control skin fibroblasts.”

May combine two claims.

Treating control fibroblasts with conditioned medium from hEDS or HSD cells disorganizes the fibronectin extracellular matrix, while their own or cEDS conditioned medium does not.

“This hypothesis was corroborated by the treatment of control fibroblasts with CM from all cell types, which induced the disorganization of the FN-ECM only in the presence of hEDS and HSD cells' CM (Fig. 2D).”

May combine two claims.

Total and nuclear Snail1/Slug are increased in hEDS and HSD cells compared with control and cEDS fibroblasts, with nuclear levels almost undetectable in control and cEDS.

“WB analyses confirmed that both total and nuclear Snail1/Slug were increased in hEDS and HSD cells compared to control and cEDS fibroblasts; in cEDS and control nuclei the transcription factor was almost undetectable (Fig. 5B).”

May combine two claims.

In hEDS and HSD cells, αvβ3 integrin in focal adhesions is phosphorylated at tyrosine 773 and co-distributes with ILK.

“Indeed, we demonstrated that the αvβ3 integrin, localized in focal adhesions of hEDS and HSD cells, is phosphorylated in tyrosine 773 and co-distributes with ILK”

• cites [10] sourceNot checked yet

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The quote only partly states this claim.

Control fibroblasts grown with their own or cEDS conditioned medium do not recruit αvβ3 integrin, as under basal conditions.

“Control fibroblasts, grown in the presence of their own CM and of that recovered from cEDS cells, did not recruit the αvβ3 integrin as well as under basal conditions.”

May combine two claims.

Control fibroblasts treated with hEDS/HSD conditioned medium redistribute ILK to focal adhesions, recruit αvβ3 integrin, organize α-SMA stress fibres, and acquire a myofibroblast-like phenotype like hEDS/HSD cells.

“This is confirmed by the fact that control fibroblasts, after treatment with the CM from hEDS and HSD cells, redistributed ILK in focal adhesions together with the concomitant recruitment of αvβ3 integrin, organized α-SMA in stress fibres and acquired a myofibroblastlike phenotype like that of hEDS and HSD cells.”

May combine two claims.

ILK inhibition in hEDS/HSD cells disorganizes the α-SMA cytoskeleton and decreases αvβ3 integrin in these structures.

“the acquisition of the myofibroblast-like phenotype was obtained by the disorganization of the α-SMA cytoskeleton observed after ILK inhibition that was associated with the gradual decrease of the αvβ3 integrin in these structures.”

May combine two claims.

In hEDS and HSD cell media, the 82 kDa active MMP-9 form is abundant, while control fibroblasts secrete only the inactive 95 kDa proenzyme.

“In hEDS and HSD cells media the MMP-9 82 kDa active form was present at high levels, whereas only the inactive 95 kDa proenzyme was secreted by control fibroblasts, suggesting an increased proteolytic activity of this protease in hEDS and HSD media.”

May combine two claims.

Paxillin organization is unchanged after hEDS or HSD conditioned medium treatment, indicating fibrillar sites are maintained.

“The organization of paxillin, a marker of both fibrillar and focal contacts, did not show any difference after treatment with the hEDS and HSD cells' CM, indicating that fibrillar sites are maintained in the presence of CM.”

May combine two claims.

In cEDS cells, MMP-9 protein is nearly undetectable, matching about threefold lower mRNA levels than control cells.

“In cEDS cells, the MMP-9 protein was almost undetectable, consistent with the 3-fold lower mRNA levels detected by qPCR compared to control cells (Fig. 2D).”

May combine two claims.

ILK localizes in focal and fibrillar adhesions in control and cEDS fibroblasts, but mainly in focal adhesions in hEDS and HSD cells.

“In control, cEDS, hEDS and HSD cells ILK was detected by IF with a different localization, i.e., in focal and fibrillar adhesions in control and cEDS fibroblasts and mainly in focal adhesions in hEDS and HSD cells (Fig. 3B).”

May combine two claims.

Control fibroblasts do not express αvβ3 integrin under standard growth conditions but organize it preferentially in focal adhesions with hEDS/HSD conditioned medium.

“Furthermore, the evidence that control fibroblasts, which do not express this integrin in standard growth conditions ( [9,10], this work), organize it preferentially in focal adhesions in the presence of hEDS and HSD CM corroborates the involvement of αvβ3 integrin in focal contacts in the transdifferentiation of fibroblasts into myofibroblasts.”

• cites [9] sourceNot checked yet

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• cites [10] sourceNot checked yet

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May combine two claims.

In hEDS and HSD cells, Cpd22 up to 2.0 μM progressively reduces αvβ3 integrin in fibrillar and focal adhesions and disassembles the α-SMA cytoskeleton, with detachment at 4.0 μM.

“In hEDS and HSD cells, the treatment with low amounts of Cpd22 (up to 2.0 μM) was associated both with the progressive reduction of αvβ3 integrin in fibrillar and focal adhesions and α-SMA cytoskeleton disassembly (Fig. 4), finally leading to cell detachment at 4.0 μM (not shown).”

May combine two claims.

ILK inhibition with Cpd22 in hEDS/HSD cells disorganizes the α-SMA cytoskeleton and significantly reduces nuclear Snail1/Slug accumulation.

“As a further evidence, the inhibition of ILK with Cpd22 in hEDS and HSD cells not only led to the disorganization of the α-SMA cytoskeleton but also significantly reduced the nuclear accumulation of Snail1/Slug.”

May combine two claims.

Control fibroblasts grown with hEDS/HSD conditioned medium disassemble their abundant fibronectin ECM scaffold and organize α-SMA cytoskeleton.

“Indeed, when control fibroblasts are grown in the presence of CM of hEDS and HSD cells their abundant FN-ECM scaffold is disassembled and the α-SMA cytoskeleton is organized.”

May combine two claims.

In control fibroblasts, Cpd22 up to 1 μM induces ILK localization in focal adhesions, αvβ3 integrin organization there, and α-SMA stress fibre organization.

“In control fibroblasts, the treatment with increasing Cpd22 concentrations (up to 1 μM) induced the progressive localization of ILK in the focal adhesions and the concomitant organization of the αvβ3 integrin in these contacts along with the organization of α-SMA stress fibres (Fig. 4).”

May combine two claims.

Both hEDS and HSD fibroblasts display an in vitro myofibroblast-like phenotype with increased migratory ability, which sets them apart from cEDS and vEDS cells.

“Overall, these data demonstrate that both hEDS and HSD fibroblasts exhibit in vitro a myofibroblast-like phenotype with an enhanced migratory capability, which distinguishes them from cEDS and vEDS cells.”

• AssumptionRests on an assumption: The hEDS and HSD patient cell strains used are representative of the broader hEDS and HSD patient populations.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The 2017 EDS nosology criteria for hEDS are more stringent than the earlier Villefranche criteria for EDS hypermobility type and the Brighton criteria for joint hypermobility syndrome.

“This new set of clinical criteria is stricter than the previous Villefranche criteria, used for the diagnosis of EDS hypermobility type (EDS-HT) [2], and the Brighton criteria for joint hypermobility syndrome (JHS) [3].”

• cites [2] sourceNot checked yet

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• cites [3]Not checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Myofibroblasts produce and release cytokines, extracellular matrix molecules such as collagens and fibronectin, and matrix metalloproteinases that regulate ECM turnover and remodeling.

“Myofibroblasts also synthesize and secrete cytokines, ECM molecules, such as COLLs and FN, and matrix metalloproteinases (MMPs) that control the ECM turnover and remodeling [22,23].”

• cites [22] sourceNot checked yet

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• cites [23] sourceNot checked yet

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May combine two claims.

In skin fibroblasts from hEDS and HSD patients, there is extensive disorganization of extracellular matrix structural components and altered expression of genes involved in ECM organization, cell adhesion, and immune and inflammatory responses.

“In hEDS and HSD patients' skin fibroblasts, we previously reported a widespread disarray of several extracellular matrix (ECM) structural components, such as type I collagen, COLLIII, and COLLV, fibronectin (FN), fibrillins, tenascins, and elastin, together with a dysregulated expression of many genes related to ECM organization, cell adhesion, immune and inflammatory responses [8].”

• cites [8] sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

The label hypermobility spectrum disorders (HSD) was created for people with symptomatic joint hypermobility who do not satisfy the 2017 hEDS criteria.

“For all those phenotypes presenting symptomatic JHM plus one or more of its secondary manifestations, but that do not fulfill the new hEDS criteria, the term hypermobility spectrum disorder(s) (HSD) was introduced to not leave patients with an incomplete clinical presentation without a defined diagnosis [4].”

• cites [4] sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Hypermobile Ehlers-Danlos syndrome is an inherited connective tissue disorder whose molecular cause is unknown, and it is primarily marked by widespread joint hypermobility, joint instability complications, and minor skin changes.

“Hypermobile Ehlers-Danlos syndrome (hEDS) is a heritable connective tissue disorder with unknown molecular basis mainly characterized by generalized joint hypermobility, joint instability complications, and minor skin changes.”

• Also stated in: § 1. Introduction · plain

  “Hypermobile EDS (hEDS), a dominantly inherited trait without a defined molecular basis, is mainly defined by gJHM, joint dislocations/instability complications, i.e., musculoskeletal chronic pain, and a combination of mucocutaneous features [1].”

• cites [1] sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

During fibroblast-to-myofibroblast transition, N-cadherin levels fall while OB-cadherin/cadherin-11 levels rise.

“indeed, during the fibroblast-tomyofibroblast transition a decrease of N-cadherin expression and an increase of OB-cadherin/cadherin-11 have been described [21].”

• cites [21] sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

CCN2/CTGF and CCN1/CYR61 respectively stimulate and antagonize myofibroblast proliferation and differentiation and the resolution of inflammation.

“Furthermore, during tissue repair, growth factors are induced by tissue injury and inflammation including CCN2/CTGF and CCN1/CYR61 that have respectively a stimulatory and antagonist effect on myofibroblasts proliferation and differentiation and on the resolution phase of the inflammatory state [24,25].”

• cites [24] sourceNot checked yet

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• cites [25] sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The transition from fibroblast to myofibroblast includes production of α-smooth muscle actin and its assembly into stress fibres.

“Phenotypic changes occurring in fibroblast-to-myofibroblast transition include the expression of the α-smooth muscle actin (α-SMA) and its incorporation into stress fibres that allows cell's migration and contraction [19,20].”

• cites [19] sourceNot checked yet

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• cites [20] sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Myofibroblasts or myofibroblast-like contractile cells have been found in several inflamed tissues, including ligament, meniscus, and tendon synovia.

“Presence of myofibroblasts, or myofibroblast-like contractile cells, has been demonstrated in several tissues during inflammation including synovia of ligaments, menisci, and tendons [27].”

• cites [27] sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Quantitative PCR reactions were performed in triplicate.

“All qPCR reactions were run in triplicate.”

May combine two claims.

Fibronectin and possibly other ECM fragments might act as DAMPs in hEDS and HSD cells, likely contributing to an in vitro inflammation-like phenotype that could relate to chronic pain.

“Fragments of FN and possibly of other ECM proteins might act as DAMPs in hEDS and HSD cells, likely contributing to the generation of the in vitro inflammation-like phenotype, which could be related to the chronic pain observed in hEDS and HSD patients [49].”

• cites [49] sourceNot checked yet

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Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing).

hEDS and HSD patients share many characteristics with CID patients.

“All these data allow to hypothesize that hEDS and HSD patients share many characteristics with those affected by CID [7].”

• cites [7]Not checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Migration assay results are reported as means ± SEM from three independent experiments each run in triplicate.

“Data are expressed as means ± SEM from 3 independent experiments performed in triplicate.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Every patient received their diagnosis at the Centre of Heritable Connective Tissue Disorders and EDS of the University Hospital Spedali Civili of Brescia.

“All patients were diagnosed at the Centre of Heritable Connective tissue disorders and EDS of the University Hospital Spedali Civili of Brescia.”

May combine two claims.

hEDS and HSD are likely not distinct entities but part of a phenotypic continuum with common altered tissue homeostasis and chronic inflammatory condition.

“Our findings suggest that hEDS and HSD are likely not distinct entities, but rather part of a phenotypic continuum characterized by a common altered tissue homeostasis and a chronic inflammatory condition.”

• AssumptionRests on an assumption: The hEDS/HSD cell strains studied are representative of the broader hEDS/HSD patient population.

Classified as probably not a substantial claim.

The images are representative of multiple cell strains: 2 controls, 4 cEDS, 4 vEDS, 4 hEDS, and 3 HSD.

“The images represent cell strains analyzed at 5 th in vitro passage and are representative of 2 controls, 4 cEDS, 4 vEDS, 4 hEDS, and 3 HSD cell strains (see Supplementary Fig. 1).”

Probably not a substantial claim: may not pass S3 (load-bearing).

Statistical significance between EDS and control cells was assessed with an unpaired Student's t-test.

“Statistical significance between EDS compared to control cells was determined using an unpaired Student's t-test with the GraphPad Prism software”

May combine two claims.

hEDS and HSD cell media promote ILK recruitment to focal adhesions through interaction with αvβ3 integrin, which clusters there only with hEDS/HSD conditioned medium.

“Therefore, it is likely that the hEDS and HSD cells' media promote the ILK recruitment in focal adhesions through its interaction with the αvβ3 integrin, preferentially clustered in these sites only in the presence of hEDS and HSD cells' CM (Fig. 3A,D).”

Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

The study received approval from the local Ethical Committee 'Comitato Etico di Brescia, ASST Spedali Civili, Brescia, Italia' (registration numbers NP2378 and NP2658).

“following approval by the local Ethical Committee”

Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing).

Snail1/Slug involvement in upregulated MMP-9 expression in the hEDS/HSD fibroblast-to-myofibroblast transition can be hypothesized.

“Therefore, the involvement of Snail1/Slug in the upregulated expression of MMP-9, acting in the hEDS and HSD fibroblastto-myofibroblast transition, can be hypothesized.”

May combine two claims.

In focal contacts of hEDS and HSD cells, the αvβ3 integrin-ILK complex is specifically involved in establishing the myofibroblast-like phenotype because β1 integrin subunits are undetectable.

“In the focal contacts of hEDS and HSD cells, the αvβ3 integrin-ILK complex should be specifically involved in the establishment of the myofibroblast-like phenotype, because of the undetectable organization of β1 integrin subunits in these cells.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The hEDS/HSD phenotype was observed at different in vitro passages (1st-10th) with similar results.

“The cells were analyzed at different in vitro cell growth passages (1 st -10 th ) with similar results (not shown).”

May combine two claims.

A myofibroblast-like phenotype identified in dermal cells from hEDS and HSD patients distinguishes them from cEDS and vEDS fibroblasts, suggesting a common inflammatory-like state consistent with systemic clinical manifestations.

“In conclusion, this study reports the identification of a myofibroblast-like phenotype in dermal cells isolated both from hEDS and HSD patients, which distinguishes them from cEDS and vEDS fibroblasts, suggesting the presence of a common inflammatory-like state consistent with the patients' systemic clinical manifestations.”

• AssumptionRests on an assumption: The hEDS/HSD cell strains studied are representative of the broader hEDS/HSD patient population.

Probably not a substantial claim: may not pass S3 (load-bearing).

All immunofluorescence experiments were repeated three times.

“All experiments were repeated three times.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Each Western blot experiment was performed twice.

“Each experiment was performed two times.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The hEDS and HSD patients were reclassified according to the 2017 EDS nosology.

“which were re-classified following the 2017 EDS nosology [1]”

• cites [1] sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

qPCR was performed in triplicate and results expressed as mean ± SEM.

“qPCR was performed in triplicate, and the results are expressed as mean ± SEM (*P < 0.05, **P < 0.01).”

Probably not a substantial claim: may not pass S3 (load-bearing).

The purity of the nuclear cell fraction was verified by immunoreacting nuclear extracts with an anti-β actin antibody.

“The nuclear extracts were also immunoreacted with the anti-β actin mAb to verify the purity of the cell fraction.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The images in Fig. 5C are representative of two hEDS and two HSD cell strains.

“The images are representative of 2 hEDS, and 2 HSD cell strains (not shown).”

Probably not a substantial claim: may not pass S3 (load-bearing).

The migration data are presented as means ± SEM from three independent experiments, each performed in triplicate.

“Data represent the means ± SEM from 3 independent experiments performed in triplicate for the above groups”

Probably not a substantial claim: may not pass S3 (load-bearing).

Quantitative PCR was performed in triplicate.

“qPCR was performed in triplicate, and the results are expressed as mean ± SEM”

Probably not a substantial claim: may not pass S3 (load-bearing).

The immunofluorescence images shown are typical of multiple cell strains: two control, four cEDS, two hEDS, and two HSD.

“The images are representative of 2 control, 4 cEDS, 2 hEDS (P1, P2), and 2 HSD (P5, P6) cell strains (not shown).”

• Also stated in: § 4. Discussion · plain

  “The images are representative of 2 control, 4 cEDS, 2 hEDS, and 2 HSD cell strains (not shown).”

• Also stated in: § 4. Discussion · plain

  “The images are representative of 2 control, 4 cEDS, 2 hEDS, and 2 HSD cell strains (data not shown).”