The C5a/C5a receptor 1 axis controls tissue neovascularization through CXCL4 release from platelets

Platelets contribute to the regulation of tissue neovascularization, although the specific factors underlying this function are unknown. Here, we identified the complement anaphylatoxin C5a-mediated activation of C5a receptor 1 (C5aR1) on platelets as a negative regulatory mechanism of vessel formation. We showed that platelets expressing C5aR1 exert an inhibitory effect on endothelial cell functions such as migration and 2D and 3D tube formation. Growth factor- and hypoxia-driven vascularization was markedly increased in C5ar1−/− mice. Platelet-specific deletion of C5aR1 resulted in a proangiogenic phenotype with increased collateralization, capillarization and improved pericyte coverage. Mechanistically, we found that C5a induced preferential release of CXC chemokine ligand 4 (CXCL4, PF4) from platelets as an important antiangiogenic paracrine effector molecule. Interfering with the C5aR1-CXCL4 axis reversed the antiangiogenic effect of platelets both in vitro and in vivo. In conclusion, we identified a mechanism for the control of tissue neovascularization through C5a/C5aR1 axis activation in platelets and subsequent induction of the antiangiogenic factor CXCL4.


Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.
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A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

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For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above. All studies must disclose on these points even when the disclosure is negative.

Sample size
Sample sizes of the hindlimb ischemia experiments were calculated with G*Power (freeware, Faul 2007). For all other experiments, no statistical methods were used to predetermine sample size. Where appropriate, all experiments were performed at least three times. Multiple animals and/or biological samples were measured in all assays, as is consistent with these types of studies.
Data exclusions No data were excluded from analysis.

Replication
All experiments were performed as technical or biological replicates as appropriate for the experimental design and setup. The n-number is stated in the figure legends. All data are reported.
Randomization No randomization occured. Due to small n-numbers randomization was not considered paramount for the overall quality of data. However, much care was given to comparability.

Blinding
During data collection and analysis, blinding to group allocations was performed at every instance for all experiments. During performance of experiments, blinding was not possible to ensure experimental accuracy and reduce potential sources of experementator error. Specifically, animal experiments with heterogenic mouse litters (knockout-animals and littermate controls) were performed in a blinded fashion. Qualitative readout of microCT images was performed in an experimentor-blinded fashion. Image analyses were performed in an experimentor-blinded fashion.

Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. Validation For flow cytometry experiments: All antibodies used were recommended by the manufacturer for flow cytometry applications. The required concentration was arrived at by titration experiments with maximization of the calculated stain index. Specificity was tested both by fluorescence-minus-one controls as well as isotype control staining. For immunofluorescence staining: All antibodies used were recommended by the manufacturer for application in immunofluorescent staining of frozen sections. Specificity was assessed by performing staining with and without primary antibody but with secondary antibody on the same slides as a standard control. For initial testing of primary antibodies, we used isotype controls in equimolar concentrations. All secondary antibodies were approved by the manufacturer for the intended use.

Eukaryotic cell lines Policy information about cell lines
Cell line source(s) MHEC-5T (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures ), HUVECs (PromoCell, Heidelberg, Germany)

Authentication
No authentication was performed.

Mycoplasma contamination
The cell lines were not tested.

Commonly misidentified lines (See ICLAC register)
None of the cell lines used is a commonly misidentified line.

Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research

Laboratory animals
A detailed description of each mouse, strain, sex and age is reported in the materials and methods section of the manuscript. Mice for hindlimb ischemia experiments were aged 10-12 weeks, mice for blood withdrawal were 6-16 weeks. Light cycle was 12 h, temperature 20-22 °C, humidity 40-60%.

Wild animals
The study did not involve wild animals Field-collected samples The study did not include field-collected samples

Ethics oversight
All animal procedures were approved by the regional animal care and use committee of the District of Tübingen, Baden-Württemberg (Konrad-Adenauer-Straße 20, 72072 Tübingen, Germany). All animal experiments were performed in accordance with the German law guidelines of animal care.

nature research | reporting summary
April 2020 Note that full information on the approval of the study protocol must also be provided in the manuscript.

Human research participants
Policy information about studies involving human research participants

Population characteristics
Only very few healthy volunteers participated in the experiments reported here by donating blood for human platelets isolation. All participants were free from drug usage of any kind for 2 weeks. Both females and males were included. Only healthy individuals donated blood. Thus, there were no diagnoses, the mean age of research participants was 31.42 +/-4.1 (SEM) Recruitment Volunteers participated freely. No further data was collected on them. As only cells were isolated from the blood of volunteers and cells from each individual were used for all groups in the subsequent experiments, self-selection bias does not apply.

Ethics oversight
All procedures were in accordance with German Federal Law and Regulations and performed under supervision of the institutional ethics committee. For experiments with human material, written informed consent was received from participants prior to inclusion in the study. The study was approved by the institutional ethics committee (270/2011BO1) and complies with the Declaration of Helsinki and the good clinical practice guidelines.
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
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All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Instrument
A Fortessa Instrument manufactured by BD Biosciences was used for analyzing some samples. Some samples were analyzed on a FACS-Calibur flow cytometer (Becton-Dickinson, Heidelberg, Germany). Most samples were analyzed on a Cytoflex-S (4 lasers, 13 colours, Beckman Coulter).
Software FACS Diva (BD Biosciences) software was used for data collection from all biological samples run on the Fortessa and the Calibur Instrument (BD Biosciences). Data analysis was performed using FlowJo v9. For the Cytoflex, CytExpert Softwatre (v. 2. 4) was used for acquisition and analysis.

Cell population abundance
The abundance of platelets was about 7 -10 % relative to all events in murine whole blood. The abundance of single platelets in human whole blood was about 3-6 % relative to all events. The puritiy of isolated platelets was over 95% both for human as well as mouse samples.

Gating strategy
Gating strategies are shown on representative flow cytometry plots where necessary.
Gating for mouse as well as human platelets was performed by CD41 and CD45 as well as FSC and SSC at a logarithmic scale. Platelets were defined as CD45-CD41+ and differentiated from debris using FSC and SSC (see also Suppl. Fig. 26).
Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.