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Research
Sang Han
Universidade Federal de Sao Paulo Escola Paulista de Medicina
Corresponding Author
ORCiD: https://orcid.org/0000-0002-4953-7680
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background
Peripheral arterial disease (PAD) affects millions of people and compromises quality of life. Critical limb ischemia (CLI), which is the most advanced stage of PAD, can cause nonhealing ulcers and strong chronic pain, and it shortens the patients’ life expectancy. Cell-based angiogenic therapies are becoming a real therapeutic approach to treat CLI. Pericytes are cells that surround vascular endothelial cells to reinforce vessel integrity and regulate local blood pressure and metabolism. In the past decade, researchers also found that pericytes may function as stem or progenitor cells in the body, showing the potential to differentiate into several cell types. We investigated the gene expression profiles of pericytes during the early stages of limb ischemia, as well as the alterations in pericyte subpopulations to better understand the behavior of pericytes under ischemic conditions.
Methods
In this study, we used a hindlimb ischemia model to mimic CLI in C57/BL6 mice and explore the role of pericytes in regeneration. To this end, muscle pericytes were isolated at different time points after the induction of ischemia. The phenotypes and transcriptomic profiles of the pericytes isolated at these discrete time points were assessed using flow cytometry and RNA sequencing.
Results
Ischemia triggered proliferation and migration and upregulated the expression of myogenesis-related transcripts in pericytes. Furthermore, the transcriptomic analysis also revealed that pericytes induce or upregulate the expression of a number of cytokines with effects on endothelial cells, leukocyte chemoattraction, or the activation of inflammatory cells.
Conclusions
Our findings provide a database that will improve our understanding of skeletal muscle pericyte biology under ischemic conditions, which may be useful for the development of novel pericyte-based cell and gene therapies.
Keywords
peripheral arterial disease, limb ischemia, muscle, pericytes, RNA-seq
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
This is a list of supplementary files associated with this preprint. Click to download.
- Additionalfile1.xlsx
- Supplementaryfigure1lowercase.tiff
Event numbers of endothelial cell and pericyte populations. (a-d) Event numbers of (a) endothelial cells, (b) pericytes, (c) type I pericytes and (d) type II pericytes in one cell suspension sample prepared from one ischemic gastrocnemius muscle. Student’s t test was used to perform statistical analyses. *, p<0.05; **, p<0.01
- Supplementaryfigure2lowercase.tiff
Isolation of pericytes (CD45-CD31-CD146+) using FACS. Representative figure of gated pericytes (CD146+CD31-) (P5) in the CD45- population processed using FACS (upper panel). Then, the purity of the sorted pericytes was analyzed again using the same gating criteria (lower panel).
- Supplementaryfigure3lowercase.tiff
Comparison of enriched KEGG pathways between dynamic profiles. The results of analyses and comparisons of the enriched KEGG pathways in each dynamic profile determined by STEM using ClusterProfiler packages.
- Supplementaryfigure4lowercasev2.tiff
Heatmaps of the expression levels of genes in cell-cell contact and extracellular matrix categories based on GO terms. Heatmaps present the expression profiles of genes involved in (a) cell junction regulation, (b) ECM regulation, and (c) regulation of cell adhesion that were selected based on the GO tree (see Table 1). The genes mentioned in the text are marked with arrows.
- Supplementaryfigure5lowercasev2.tiff
Cytokine production in pericytes. Heatmaps present the expression profiles of genes involved in (a) cytokine production and (b) leukocyte activation that were selected based on the GO tree (see Table 1). (c) Quantitation of the CD45+ hematopoietic cell population in ischemic muscles. The genes mentioned in the text are marked with arrows.
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A new preprint design is coming!
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See the published version of this preprint at Stem Cell Research & Therapy.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research
Sang Han
Universidade Federal de Sao Paulo Escola Paulista de Medicina
Corresponding Author
ORCiD: https://orcid.org/0000-0002-4953-7680
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
View the published article at Stem Cell Research & Therapy.
Version 1
Posted 21 Dec, 2020
No community comments so far
Editorial decision: Major Revision
On 03 Jan, 2021
Reviewer #4 agreed
On 02 Jan, 2021
Review #1 received
Received 01 Jan, 2021
Reviewer #3 agreed
On 31 Dec, 2020
Review #2 received
Received 29 Dec, 2020
Reviewer #2 agreed
On 21 Dec, 2020
Reviewer #1 agreed
On 21 Dec, 2020
Reviewers invited
Invitations sent on 19 Dec, 2020
Editor assigned
On 15 Dec, 2020
Submission checks complete
On 15 Dec, 2020
Editor invited
On 15 Dec, 2020
First submitted
On 12 Dec, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at Stem Cell Research & Therapy.
Background
Peripheral arterial disease (PAD) affects millions of people and compromises quality of life. Critical limb ischemia (CLI), which is the most advanced stage of PAD, can cause nonhealing ulcers and strong chronic pain, and it shortens the patients’ life expectancy. Cell-based angiogenic therapies are becoming a real therapeutic approach to treat CLI. Pericytes are cells that surround vascular endothelial cells to reinforce vessel integrity and regulate local blood pressure and metabolism. In the past decade, researchers also found that pericytes may function as stem or progenitor cells in the body, showing the potential to differentiate into several cell types. We investigated the gene expression profiles of pericytes during the early stages of limb ischemia, as well as the alterations in pericyte subpopulations to better understand the behavior of pericytes under ischemic conditions.
Methods
In this study, we used a hindlimb ischemia model to mimic CLI in C57/BL6 mice and explore the role of pericytes in regeneration. To this end, muscle pericytes were isolated at different time points after the induction of ischemia. The phenotypes and transcriptomic profiles of the pericytes isolated at these discrete time points were assessed using flow cytometry and RNA sequencing.
Results
Ischemia triggered proliferation and migration and upregulated the expression of myogenesis-related transcripts in pericytes. Furthermore, the transcriptomic analysis also revealed that pericytes induce or upregulate the expression of a number of cytokines with effects on endothelial cells, leukocyte chemoattraction, or the activation of inflammatory cells.
Conclusions
Our findings provide a database that will improve our understanding of skeletal muscle pericyte biology under ischemic conditions, which may be useful for the development of novel pericyte-based cell and gene therapies.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
This is a list of supplementary files associated with this preprint. Click to download.
- Additionalfile1.xlsx
- Supplementaryfigure1lowercase.tiff
Event numbers of endothelial cell and pericyte populations. (a-d) Event numbers of (a) endothelial cells, (b) pericytes, (c) type I pericytes and (d) type II pericytes in one cell suspension sample prepared from one ischemic gastrocnemius muscle. Student’s t test was used to perform statistical analyses. *, p<0.05; **, p<0.01
- Supplementaryfigure2lowercase.tiff
Isolation of pericytes (CD45-CD31-CD146+) using FACS. Representative figure of gated pericytes (CD146+CD31-) (P5) in the CD45- population processed using FACS (upper panel). Then, the purity of the sorted pericytes was analyzed again using the same gating criteria (lower panel).
- Supplementaryfigure3lowercase.tiff
Comparison of enriched KEGG pathways between dynamic profiles. The results of analyses and comparisons of the enriched KEGG pathways in each dynamic profile determined by STEM using ClusterProfiler packages.
- Supplementaryfigure4lowercasev2.tiff
Heatmaps of the expression levels of genes in cell-cell contact and extracellular matrix categories based on GO terms. Heatmaps present the expression profiles of genes involved in (a) cell junction regulation, (b) ECM regulation, and (c) regulation of cell adhesion that were selected based on the GO tree (see Table 1). The genes mentioned in the text are marked with arrows.
- Supplementaryfigure5lowercasev2.tiff
Cytokine production in pericytes. Heatmaps present the expression profiles of genes involved in (a) cytokine production and (b) leukocyte activation that were selected based on the GO tree (see Table 1). (c) Quantitation of the CD45+ hematopoietic cell population in ischemic muscles. The genes mentioned in the text are marked with arrows.
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