Semaphorin3F reduces vascular endothelial and smooth muscle cell PI3K activation and decreases neointimal plaque formation
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Evaluation Summary:
The authors provide novel evidence that semaphorin signaling (SEMA3F) is engaged in the vascular endothelium and smooth muscle to confer atheroprotection. They show that SEMA3F reduces the activity of key enzyme Phosphoinositide 3-kinase to decrease smooth muscle cell proliferation, migration, and phenotype switching, which contributes to atheroprotection. The study has significant translational potential and yields a new therapeutic target.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
We previously conducted genetic analyses, and identified semaphorin signaling as associating with coronary artery disease. Of the semaphorins, human vascular expression profiling suggested SEMA3F as potentially linked to atherogenesis. In hyperlipidemic mice, SEMA3F reduced aortic lesion area, and increased fibrous cap endothelial content, leading to plaque stability. In a disturbed-flow-mediated endothelial dysfunction-driven lesion model, the absence of Sema3f increased plaques, further implicating SEMA3F in endothelial function. Monocyte adhesion to Sema3f -/- vascular endothelial cells (VECs) was elevated, driven by increased PI3K activity, leading to increased NF-κB-mediated elevation in VCAM1 and ICAM1 expression, suggesting that SEMA3F reduces VEC PI3K activity. Increased permeability led to increased monocyte transmigration through Sema3f -/- VECs, and mTOR phosphorylation was decreased, suppressing VE-cadherin expression and cell-cell adherens junction stability. Actomyosin fiber formation was decreased in Sema3f -/- VECs, which was reversed by PI3K inhibition, further implicating SEMA3F in adherens junction stability. In Sema3f -/- vascular smooth muscle cells (VSMCs), active PI3K was also increased. PI3K facilitates VSMC proliferation, migration, and pro-atherogenic phenotype switching, which were reduced by SEMA3F. In agreement, in a model of VSMC proliferation and migration-induced neointima formation, SEMA3F reduced plaques. Semaphorin3F is causally atheroprotective. SEMA3F’s suppression of VEC and VSMC PI3K activation may contribute to its atheroprotection.
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Evaluation Summary:
The authors provide novel evidence that semaphorin signaling (SEMA3F) is engaged in the vascular endothelium and smooth muscle to confer atheroprotection. They show that SEMA3F reduces the activity of key enzyme Phosphoinositide 3-kinase to decrease smooth muscle cell proliferation, migration, and phenotype switching, which contributes to atheroprotection. The study has significant translational potential and yields a new therapeutic target.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
In this manuscript, the authors proposed that SEMA3F inhibits PI3K activity in vascular endothelial and smooth muscle cells to provide an atheroprotective effect. The authors used a combination of cell culture and in vivo studies to provide evidence that loss of SEMA3F increases PI3K signaling. The authors performed detailed analyses to identify the involved mechanism through which SEMA3F inhibits PI3K activity. This work provides some novel directions for SEMA3F-based candidates for atheroprotection. However, there are a few limitations that need to be addressed:
1. Authors used global KOs of SEMA3F which does not allow to distinguish whether SEMA3F from endothelial or smooth muscle or macrophage is contributing to the atheroprotective effect. How about compensatory mechanism(s) activated in response to …
Reviewer #1 (Public Review):
In this manuscript, the authors proposed that SEMA3F inhibits PI3K activity in vascular endothelial and smooth muscle cells to provide an atheroprotective effect. The authors used a combination of cell culture and in vivo studies to provide evidence that loss of SEMA3F increases PI3K signaling. The authors performed detailed analyses to identify the involved mechanism through which SEMA3F inhibits PI3K activity. This work provides some novel directions for SEMA3F-based candidates for atheroprotection. However, there are a few limitations that need to be addressed:
1. Authors used global KOs of SEMA3F which does not allow to distinguish whether SEMA3F from endothelial or smooth muscle or macrophage is contributing to the atheroprotective effect. How about compensatory mechanism(s) activated in response to SEMA3F global deletion? Some discussion about it will enhance quality of work.
2. Authors used Wortmannin as a PI3K inhibition. Wortmannin is not a selective inhibitor and it also disrupts the actin cytoskeleton. The authors should use another PI3K inhibitor to confirm their findings.
3. It is unclear which class or subtype of PI3K is affected in response to SEMA3F signaling.
4. Actin blot in Figure 3D is not acceptable wand would need to be replaced. Molecular weight markers need to be included in all the representative western blots.
5. Authors did not perform any vascular functional measurements (with vessel arteriographs). The results were mostly collected from in-vitro cell culture studies along with limited in vivo measurements. This should be listed as a limitation.
6. A schematic with the proposed mechanism will enhance the readability of the work.
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Reviewer #2 (Public Review):
In the current study, the authors provide a detailed examination of the potential atheroprotective role of semaphorin 3F (SEMA3F). The semaphorin signaling pathway and other members of the semaphorin family are significantly associated with coronary heart disease in humans. Additionally, several studies have linked semaphorin proteins to atheroprotection. However, the potential causal or protective roles of SEMA3F had not been studied in atherosclerosis. Rattanasopa et al, have used a combination of chow-fed, or western-type diet(WTD)-fed Ldlr-/- mice, and Sema3f-/- mice, combined with injection of recombinant SEMA3F protein, and a partial ligation model, to study the contribution of SEMA3F in atherosclerotic plaque formation and assess changes in monocyte recruitment and intracellular signaling events. …
Reviewer #2 (Public Review):
In the current study, the authors provide a detailed examination of the potential atheroprotective role of semaphorin 3F (SEMA3F). The semaphorin signaling pathway and other members of the semaphorin family are significantly associated with coronary heart disease in humans. Additionally, several studies have linked semaphorin proteins to atheroprotection. However, the potential causal or protective roles of SEMA3F had not been studied in atherosclerosis. Rattanasopa et al, have used a combination of chow-fed, or western-type diet(WTD)-fed Ldlr-/- mice, and Sema3f-/- mice, combined with injection of recombinant SEMA3F protein, and a partial ligation model, to study the contribution of SEMA3F in atherosclerotic plaque formation and assess changes in monocyte recruitment and intracellular signaling events. These data were supported through the use of primary human coronary artery endothelial cells. The authors find that SEMA3F administration can reduce atherosclerotic lesion size and increase lesion stability in hyperlipidemic mice. Conversely, loss of SEMA3F in the Sema3f-/-, had the opposite effect in increasing lesion size and decreasing endothelial cell stability while increasing smooth muscle actin expression. Critically, loss of SEMA3F enhanced monocyte adhesion, likely due to increased VCAM1 and ICAM1 expression in endothelial cells. Additionally, SEMA3F enhanced smooth muscle cell migration and contraction. Combined, the studies provide fresh insight into the possible protective effect of SEMA3F peptide, through reduction of PI3K signaling and collagen deposition.
The conclusions of the study are generally supported by the data but would benefit from additional supportive controls, greater clarity of descriptions for imaging methodologies, and clearer image representation.
The authors show multiple important data sets that support the hypotheses, but to fully confirm the conclusions additional controls would be required. Specifically, the dissection of the signaling pathways downstream of SEMA3F and the control of mTOR, PI3K, NFkB, and the subsequent regulation of monocyte adhesion via VCAM1 or ICAM1. Limited controls were used and strong conclusions were drawn from small differences, this impacted the potential interpretation of the data
The authors use quantification of multiple histological images throughout the manuscript. Quantifying staining or fluorescent intensity to define the possible role of SEMA3F on atherosclerotic progression. There are some concerns with these experiments. Firstly, the descriptions of how plaque sections are chosen are not detailed enough in the methods. i.e how have the authors controlled for where in the plaque the sections have been collected, and how did they ensure to compare similar sections between conditions and clarity for how many individual repeats were used. Secondly, the methods are not detailed enough to fully assess how the samples were quantified. Finally, in some images (Figures 1C,1F, 2B, 3E, 5C-F, and 7A-D) it is difficult to see the difference claimed by the authors as currently presented. Combined, as these data are critical to testing the authors' hypothesis.
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