LRRC15 suppresses SARS-CoV-2 infection and controls collagen production

Although ACE2 is the primary receptor for SARS-CoV-2 infection, a systematic assessment of factors controlling SARS-CoV-2 host interactions has not been described. Here we used whole genome CRISPR activation to identify host factors controlling SARS-CoV-2 Spike binding. The top hit was a Toll-like receptor-related cell surface receptor called leucine-rich repeat-containing protein 15 (LRRC15). LRRC15 expression was sufficient to promote SARS-CoV-2 Spike binding where it forms a cell surface complex with LRRC15 but does not support infection. Instead, LRRC15 functioned as a negative receptor suppressing both pseudotyped and live SARS-CoV-2 infection. LRRC15 is expressed in collagen-producing lung myofibroblasts where it can sequester virus and reduce infection in trans. Mechanistically LRRC15 is regulated by TGF-β, where moderate LRRC15 expression drives collagen production but high levels suppress it, revealing a novel lung fibrosis feedback circuit. Overall, LRRC15 is a master regulator of SARS-CoV-2, suppressing infection and controlling collagen production associated with “long-haul” COVID-19.

Modulation by 17,20S(OH)2pD of Fibrosis-Related Mediators in Dermal Fibroblast Lines from Healthy Donors and from Patients with Systemic Sclerosis

We previously demonstrated that the non-calcemic pregnacalciferol (pD) analog 17,20S (OH)2pD suppressed TGF-β1-induced type I collagen production in cultured normal human dermal fibroblasts. In the present studies, we examined fibroblasts cultured from the lesional skin of patients with systemic sclerosis (scleroderma (SSc)) and assessed the effects of 17,20S(OH)2pD on fibrosis-related mediators. Dermal fibroblast lines were established from skin biopsies from patients with SSc and healthy controls. Fibroblasts were cultured with either 17,20S(OH)2pD or 1,25(OH)2D3 (positive control) with/without TGF-β1 stimulation and extracted for protein and/or mRNA for collagen synthesis and mediators of fibrosis (MMP-1, TIMP-1, PAI-1, BMP-7, PGES, GLI1, and GLI2). 1 7,20S(OH)2pD (similar to 1,25(OH)2D3) significantly suppressed net total collagen production in TGF-β1-stimulated normal donor fibroblast cultures and in cultures of SSc dermal fibroblasts. 17,20S(OH)2pD (similar to 1,25(OH)2D3) also increased MMP-1, BMP-7, and PGES and decreased TIMP-1 and PAI1 expression in SSc fibroblasts. Although 17,20S(OH)2pD had no effect on Gli1 or Gli2 in SSc fibroblasts, it increased Gli2 expression when cultured with TGF-β1 in normal fibroblasts. These studies demonstrated that 17,20S(OH)2pD modulates mediators of fibrosis to favor the reduction of fibrosis and may offer new noncalcemic secosteroidal therapeutic approaches for treating SSc and fibrosis.

Secretome from In Vitro Mechanically Loaded Myoblasts Induces Tenocyte Migration, Transition to a Fibroblastic Phenotype and Suppression of Collagen Production

It is known that mechanical loading of muscles increases the strength of healing tendon tissue, but the mechanism involved remains elusive. We hypothesized that the secretome from myoblasts in co-culture with tenocytes affects tenocyte migration, cell phenotype, and collagen (Col) production and that the effect is dependent on different types of mechanical loading of myoblasts. To test this, we used an in vitro indirect transwell co-culture system. Myoblasts were mechanically loaded using the FlexCell® Tension system. Tenocyte cell migration, proliferation, apoptosis, collagen production, and several tenocyte markers were measured. The secretome from myoblasts decreased the Col I/III ratio and increased the expression of tenocyte specific markers as compared with tenocytes cultured alone. The secretome from statically loaded myoblasts significantly enhanced tenocyte migration and Col I/III ratio as compared with dynamic loading and controls. In addition, the secretome from statically loaded myoblasts induced tenocytes towards a myofibroblast-like phenotype. Taken together, these results demonstrate that the secretome from statically loaded myoblasts has a profound influence on tenocytes, affecting parameters that are related to the tendon healing process.

Valsartan and Sacubitril Combination Treatment Enhances Collagen Production in Older Adult Human Skin Cells

Collagen is a major component of the skin’s support system, allowing for firmness, elasticity, and mechanical strength. In older adults, skin collagen production decreases significantly, and is associated with increased sagging, wrinkling, and thinning. The Renin Angiotensin System (RAS) is a key hormonal system that changes with age and affects multiple organ systems. While primary health benefits of Angiotensin (Ang) receptor type1 (AT1 R) blockers (ARBs) are believed to arise from systemic effects on blood pressure. There exists a skin-specific Renin Angiotensin System (RAS), but the impact of ARBs on older skin is unknown. Human skin fibroblasts from individuals aged 2 (young individual) and 57 (older individual) were treated with drugs that alter RAS: Valsartan (an ARB) and neprilysin inhibitor Sacubitril. Fibroblast proliferation and collagen production was quantified in response to the drug treatment using fluorescence microscopy. Fibroblasts from 57-year-old individuals were slower to proliferate and had less collagen content as compared to fibroblasts from young individual. Valsartan alone treatment had no effect on collagen production from young or old fibroblasts. In contrast, Sacubitril treatment increased collagen production by approximately three-fold in young (2.87 ± 0.27 RFU, P<.0001), and older (2.93 ± 0.53 RFU, P<.0001) fibroblasts. Concomitant treatment with Valsartan and Sacubitril increased collagen production by five-fold increase (5.36 ± 1.08 RFU, P<.0001) in young fibroblasts, and four-fold (4.18 ± 0.96 RFU, P=.003) in older cells. This study demonstrates a novel use for the widely prescribed drug combination, Sacubitril and Valsartan, which significantly improves collagen production in older adult fibroblasts.

LRRC15 suppresses SARS-CoV-2 infection and controls collagen production

Although ACE2 is the primary receptor for SARS-CoV-2 infection, a systematic assessment of factors controlling SARS-CoV-2 host interactions has not been described. Here we used whole genome CRISPR activation to identify host factors controlling SARS-CoV-2 Spike binding. The top hit was a Toll-like receptor-related cell surface receptor called leucine-rich repeat-containing protein 15 (LRRC15). LRRC15 expression was sufficient to promote SARS-CoV-2 Spike binding where it forms a cell surface complex with LRRC15 but does not support infection. Instead, LRRC15 functioned as a negative receptor suppressing both pseudotyped and live SARS-CoV-2 infection. LRRC15 is expressed in collagen-producing lung myofibroblasts where it can sequester virus and reduce infection in trans. Mechanistically LRRC15 is regulated by TGF-β, where moderate LRRC15 expression drives collagen production but high levels suppress it, revealing a novel lung fibrosis feedback circuit. Overall, LRRC15 is a master regulator of SARS-CoV-2, suppressing infection and controlling collagen production associated with "long-haul" COVID-19.

Dermal extracellular matrix response to facetem implant: a randomised controlled experimental study

Background. A leading concept in modern regenerative medicine is the perspective of using own body resources to remodel organs and tissues via the formation of “living scaffold”. A pivotal role in the formation of dermal scaffold is played by fibroblasts that produce extracellular matrix (ECM).Aim. A study of the fibroblast activation mechanism mediating synthesis of the dermal ECM fibrillar component under the Facetem filler administration.Methods. The experimental trial was conducted in Wistar male rats (72 animals). Animals had 0.05 mL Facetem (Korea) injections subdermally. The filler is a calcium-containing product featuring gradual degradation delivered through structural microspheric properties of the Lattice-pore technology. Biological material was sampled at weeks 1 and 2 of months 1, 2, 3 and 5. Tissues were paraffin-embedded in standard histological assays and stained with Mallory’s trichrome, Picrosirius red in polarisation microscopy and immunohistochemistry with collagen types I, III and elastin antibodies (Abcam).Results. Collagen distribution in dermis and the filling zone suggests that collagen production occurs by week 2 of the Facetem filler placement followed by an increase in synthesised matrix volume to 4.39 ± 0.7 for collagen type I and 3.9 ± 0.2 for collagen type III (p < 0.05). The synthetic activity of fi broblasts reduces by month 3, albeit with collagen production remaining above control even by the end of month 5. Elastin synthesis also initiates by week 2 of the filler injection in dermis and grows by month 3.Conclusion. The presence of Facetem filler triggers a foreign body inflammatory response in dermis. This multifactorial process initiates with protein adsorption proceeding to dermal cell recruitment and modulation of fibroblasts and macrophages. Activation of these cell types induces neocollagenesis entailing the extracellular matrix synthesis and expansion in dermis.