The experiments using primary human umbilical vein ECs (HUVECs) were approved by the Ethics Committee of the Chest Hospital of Henan Province (Zhengzhou, China). Primary HUVECs (cat. no. 8000) were obtained from ScienCell Research Laboratories, Inc., and cultured in RPMI-1640 medium supplemented with 10% FBS (both from Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1% penicillin and streptomycin in a 5% CO${}_2$ incubator. All cell lines were validated by STR profiling and tested negative for mycoplasma. Cells were all cultured in a humidified incubator at 37 °C and 5% CO${}_2$. Human CEP135 was cloned from a human cDNA library constructed using a SuperScript VILO™ cDNA kit (cat. no. 11754050; Thermo Fisher Scientific, Inc.). The RNA used for cDNA library construction was isolated from HUVECs. The CEP135 sequence was subcloned into pcDNA3.1 vectors (pcDNA3.1-Flag-CEP135; Addgene, Inc., Watertown, MA, USA) in our laboratory. The primers used for CEP135 cloning were as follows: forward 5${}^{\prime }$-CAAAATTATCTGCTGTGAAAGCTG-3${}^{\prime }$ and reverse 5${}^{\prime }$-CCAAAGCAACTGACAGTCG-3${}^{\prime }$. Plasmids were transfected into cells using Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturers’ instructions. Human CEP135 and control siRNAs were synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China) and were transfected into HUVECs using Lipofectamine RNAiMAX reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The sequences were as follows: siCEP135#1, 5${}^{\prime }$-UUUACAAGGAGUUCAUCACUCAGUC-3${}^{\prime }$; siCEP135#2, 5${}^{\prime }$-AUAACUUGUAGAGCAAGAUCUUCGC-3${}^{\prime }$; siControl (scrambled siRNA), 5${}^{\prime }$-UUAGGCGUACCUGACCAUGGAUUUC-3${}^{\prime }$.

The mouse CEP135 and control siRNAs were also synthesized by Guangzhou RiboBio Co., Ltd. and used to transfect mice during the in vivo angiogenesis assays. The sequences were as follows: siCEP135, 5${}^{\prime }$-CCAGCUGGGCUACCGCCAGA-3${}^{\prime }$; siControl (scrambled siRNA), 5${}^{\prime }$-AUUGCGUUACUAGGAUUUGGAUAUG-3${}^{\prime }$.

Antibodies against $\gamma$-tubulin (cat. no. A302-630A) were purchased from Thermo Fisher Scientific, Inc., against $\alpha$-tubulin (cat. no. sc-8035) were obtained from Santa Cruz Biotechnology, Inc., and against CEP135 (cat. no. ab75005), CDK4 (cat. no. ab108357) and cyclin D1 (cat. no. ab16663) were purchased from Abcam (Cambridge, UK). FITC- or TRITsC-conjugated secondary antibodies (cat. nos. ab6717, ab6718, ab6785 and ab6786) were obtained from Abcam. DAPI and Cell Counting Kit-8 (CCK-8) were purchased from Millipore Sigma (Burlington, MA, USA). MTT was purchased from Sangon Biotech Co. Ltd (Shanghai, China).

The experiment was performed according to a previous study [18]. All animal procedures were approved by the Institutional Animal Care and Use Committee of the Chest Hospital of Henan Province (approval no. IRM-DWLL-2020172). A total of eight female nude mice (age, 8 weeks; weight, 22–24 g; n = 4 mice/group) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The experiment was performed according to a previous study [19]. The mice were sacrificed by cervical dislocation, and the lack of heartbeat was confirmed to validate death. The mice were monitored once a day before the experiment began and twice a day until the experiment ended.

Angiogenic capacity in mice was quantified with an in vivo angiogenesis assay kit (Trevigen, Inc., Gaithersburg, MD, USA). Angioreactors were filled with a small amount (20 µL) of basement membrane preparation premixed with angiogenic factors and filled with VEGF, heparin, and fibroblast growth factor-2 (FGF-2). Positive (only contains these factors) and negative controls (without these factors and siRNAs) were also set. The experimental groups included these factors and siRNAs. Mouse siRNAs at a final concentration of 2 µmol/L were then added to the angioreactors, and the transfection was finished 11 days after the transplant. The angioreactors were incubated for 1 h at 37 °C to induce gel formation and then implanted into the dorsal flank of 8-week-old nude mice. After 11 days, all of the mice were sacrificed by cervical dislocation, and the lack of heartbeat was confirmed to validate death. The angioreactors were ~5 mm long, and the mice had no adverse reactions after implantation. Cells were recovered from the angioreactors and stained with FITC-lectin at room temperature for 30 min, which stains EC glycoproteins. The relative fluorescence intensity was measured. The mRNA levels and density were calculated according to the average value of the 4 nude mice in each group.

TRIzol® (cat. no. 15596026; Invitrogen; Thermo Fisher Scientific, Inc.) reagent was used to isolate RNA from HUVECs. Total RNA was reverse transcribed into cDNA at 42 °C for 1 h using M-MLV reverse transcriptase (cat. no. M1701; Promega Corporation, Madison, WI, USA). The 2${}^{-\mathrm{\Delta }\mathrm{\Delta }\text{Ct}}$ method was used to quantify the results [20]. The qPCR primers were designed by Primer 5.0 software (PREMIER Biosoft, Palo Alto, CA, USA), and the sequences were as follows: GAPDH, forward 5${}^{\prime }$-CCAATGTGTCCGTCGTGGAT-3${}^{\prime }$ and reverse 5${}^{\prime }$-TGAAGTCGCAGGAGACAACC-3${}^{\prime }$; CEP135, forward 5${}^{\prime }$-TACTCCGCTCGGGAAAAACC-3${}^{\prime }$ and reverse 5${}^{\prime }$-TGCGCTGAAGTTCATCCCTT-3${}^{\prime }$.

HUVECs were lysed in a buffer containing 1% Triton X-100, 150 mM NaCl and 50 mM Tris (pH 7.5). Proteins were then transferred onto polyvinylidene difluoride membranes (Millipore Sigma), which were blocked at room temperature for 2 h in Tris-buffered saline containing 0.2% Tween 20 and 5% nonfat milk. The membranes were then incubated with antibodies against CEP135 (1:500; cat. no. ab75005; Abcam), CDK4 (1:500; cat. no. ab108357; Abcam), cyclin D1 (1:500; cat. no. ab16663; Abcam) and $\beta$-actin (1:2000; cat. no. ab8226; Abcam) for 2 h and with HRP-labeled secondary antibodies (cat. nos. ab6721 and ab6728, 1:3000; Abcam) for 45 min. Proteins were visualized using an enhanced chemiluminescence detection reagent (Pierce; Thermo Fisher Scientific, Inc.) and were analyzed.

HUVECs (10${}^5$ per well) were plated onto 24-well plates precoated with Matrigel (1:1 diluted with serum-free medium) at 37 °C for 30 min. Images were captured after 3 and 6 h using an Axio Observer light microscope (Carl Zeiss AG). The tube length was measured using ImageJ 9.0 software (National Institutes of Health, Bethesda, MD, USA), and the node number was measured manually per field. The average tube length and node number were then calculated.

For immunofluorescence microscopy, 10${}^5$ HUVECs were grown on coverslips, fixed with 100% methanol at –20 °C for 20 min and blocked with 2% BSA (Millipore Sigma) in PBS for 20 min. Cells were incubated with the anti-$\alpha$-tubulin and $\gamma$-tubulin antibodies (1:1000) for 2 h and then with FITC- or TRITC-labeled secondary antibodies (1:1000) for 2 h. DAPI was used for DNA staining for 3 min. Coverslips were then examined using a microscope and analyzed by ImageJ 9.0 software. The intensity of MT was measured by ImageJ software. We quantified MT fluorescence intensity per cell, measured 10 cells in each group, and took the average value.

HUVECs were added to 96-well plates (1000 cells/well) and maintained for 48 h at 37 °C. Cells were subsequently treated with MTT agent for 4 h at 37 °C and washed with PBS. Formazan was then dissolved using 200 µL DMSO.

HUVECs were plated in 96-well plates to a density of 1000 cells/well. Cells were then treated with CCK-8 for 4 h at 37 °C and measured at 450 nm wavelength.

HUVECs transfected with siRNAs for 48 h were plated onto glass coverslips until 100% confluence. A 10-µL pipette tip was used to create a scratch, after which cells were washed twice with phosphate buffer saline (PBS) to remove cell debris and cultured in serum-free media for another 24 h. Images of the wound were captured using a light microscope at 0 and 24 h to determine the extent of wound closure using ImageJ 9.0 software, and the wound healing percentage was calculated as follows: Healing area/total area.

The proportion of HUVECs (10${}^6$) in different phases of the cell cycle (including G${}_1$, S and G${}_2$/M) was analyzed by flow cytometry. Briefly, 70% ethyl alcohol was used to fix HUVECs for 24 h at –20 °C. Subsequently, RNase A (10 mg/mL; cat. no. ST578; Beyotime, Shanghai, China) was used to incubate the cells for 10 min at 37 °C, and the cells were then incubated with 50 µg/mL propidium iodide (Abcam) at 37 °C for 30 min. Cell cycle progression was assessed using a FACSCalibur flow cytometer and CellQuest Pro 5.1 (BD Biosciences, Inc., Franklin Lakes, NJ, USA).

BD Falcon inserts (BD Biosciences, Inc., NJ, USA) were used as upper chambers, and 24-well plates were used as lower chambers. Cell culture inserts were coated with 50 µL 20% Matrigel at 37 °C for 30 min. Subsequently, 10${}^5$ HUVECs/well transfected with siRNAs were placed in the upper chamber (serum-free medium), and complete medium was added to the lower chamber. Invaded cells on the underside were fixed with 4% paraformaldehyde for 25 min and stained with crystal violet (2%) solution for 25 min, and images were captured using a microscope.

HUVECs transfected with siRNAs for 48 h were plated and grown onto coverslips until 100% confluence. The HUVEC monolayer was scratched; 3 h after scratching, the cells were fixed with 4% paraformaldehyde for 30 min.

For tubulin stabilization assays, HUVECs were placed on ice for 0 and 30 min for depolymerization, followed by immunofluorescence detection of morphology after fixing the cells were fixed with 4% paraformaldehyde for 30 min.

Microtubules (green), centrosomes (red) and nuclei (blue) were stained with $\alpha$-tubulin and $\gamma$-tubulin antibodies (1:1000) for 2 h at room temperature and then with secondary antibodies (1:1000) for 2 h. Subsequently, slides were stained with DAPI (1:3000) for 3 min. Coverslips were mounted with 90% glycerol in PBS and then examined. Border cells with the microtubule organizing center (MTOC) situated between the nucleus and the leading edge were considered polarized using ImageJ 9.0 software.

Tubulin (5 mg/mL; Cytoskeleton, Inc., Califonia, USA) and purified His-CEP135 proteins (100 nM; Guidechem, Nanjing, China) were added to a 96-well plate on ice and then transferred to a spectrophotometer. The polymerization was monitored by measuring the absorbance (350 nm). Spontaneously assembled microtubules with or without His-CEP135 (100 nM) at 37 °C were diluted with PBS, followed by shifting the temperature from 20 °C to 4 °C, according to a previous study [21].

Data were analyzed using GraphPad 8.0 software (GraphPad Software, Inc., La Jolla, CA, USA). Error bars represent the mean $\pm$ SEM. The unpaired Student’s t test was used to determine statistical significance between two groups. One-way ANOVA followed by Tukey’s post hoc test was used for multiple comparisons. p 0.05 was considered statistically significant.

To investigate whether CEP135 is essential for angiogenesis, the present study examined the effect of CEP135 siRNAs on vascular endothelial tube formation in vitro. Two different siRNAs were used to target the CDS region of CEP135 mRNA and therefore induce the knockdown of CEP135, and both were able to effectively knockdown CEP135 expression in HUVECs (Fig. 1A,B). After plating cells for 3 or 6 h, a tubular network of interconnecting branches was observed in the control siRNA group; in contrast, fewer tubes were detected in the CEP135 siRNA groups (Fig. 1C). The accumulated tube length and node numbers at 3 h were measured as an index of angiogenesis (Fig. 1D,E). In addition, a CEP135 overexpression plasmid was transfected into HUVECs, and its efficiency was confirmed by western blotting (Supplementary Fig. 1). CEP135 overexpression had modest effects on the angiogenesis of HUVECs, with only slight changes in tube length and node numbers (Fig. 1F,G and Supplementary Fig. 2). However, overexpression of CEP135 rescued the inhibition of angiogenesis in HUVECs caused by CEP135 knockdown (Fig. 1H,I). These findings indicated that CEP135 was essential for angiogenesis.

Fig. 1.

CEP135 depletion leads to angiogenic defects in HUVECs. (A,B) Western blot analysis of CEP135 and $\beta$-actin expression in HUVECs transfected with siCEP135 or siControl for 24 h. (C) HUVECs transfected with siCEP135 or siControl were plated onto Matrigel, and images were captured after 3 and 6 h. (D) Tube lengths and (E) node numbers were measured at 3 and 6 h. (F–I) HUVECs transfected with the indicated siRNAs or plasmids were plated onto Matrigel, and images were captured after 6 h. (F,H) Tube lengths and (G,I) node numbers were measured after 6 h. (B,D,E) Data were analyzed using one-way ANOVA. (F–I) Data were analyzed using unpaired Student’s t test. Error bars indicate SEM. ${}^{*}$p 0.05, ${}^{**}$p 0.01, ${}^{***}$p 0.001, as indicated or vs. siControl. Scale bar, 300 µm. Each assay was performed 3 times. ns, not significant; CEP135, centrosomal protein 135; si/siRNA, small interfering RNA; HUVECs, human umbilical vein endothelial cells.

To verify the effects of CEP135 on HUVEC angiogenesis in vitro, the present study assessed the effect of CEP135 on angiogenesis in a mouse model using semiopen angioreactors that were filled with extracellular matrix-containing angiogenic factors implanted into 4 nude mice per group (Fig. 2A). A total of 12 days after implantation, the silencing efficiency of siCEP135 was detected by qPCR (Fig. 2B). Vascular structures were identified in the angioreactors (Fig. 2C, positive control), whereas no vascular structures were detected in angioreactors without any angiogenic factors (Fig. 2C, negative control).

Fig. 2.

CEP135 knockdown leads to angiogenic defects in vivo. (A) Representative image of an athymic nude mouse with a semiclosed angioreactor. (B) The efficiency of CEP135 knockdown was measured through quantitative PCR. (C) Vascular growth for 12 days. In siRNA experiments, siCEP135 or siControl. (D) Vascular density and (E) angiogenic responses were semiquantified by FITC-lectin staining. Error bars indicate SEM. ${}^{**}$p 0.01, ${}^{***}$p 0.001, as indicated or vs. siControl. N = 4 for each group. CEP135, centrosomal protein 135; si/siRNA, small interfering RNA; NC, negative control; PC, positive control; DIVAA, directed in vivo angiogenesis assay.

Notably, CEP135 ablation in the angioreactors markedly inhibited new vascular growth induced by heparin, VEGF and FGF-2, whereas control siRNA had no effect on vascular growth into the angioreactors (Fig. 2C). To examine the angiogenic level in vivo, the vascular density was assessed and was shown to be significantly decreased in the CEP135 siRNA-treated group (Fig. 2D). Subsequently, the cells were removed from the angioreactors and stained with FITC-lectin, an EC marker. As shown in Fig. 2E, the fluorescence intensity of cells in the siCEP135 group was significantly decreased compared with the control (p 0.05), suggesting that CEP135 depletion in vivo could markedly inhibit angiogenesis. These findings confirmed an important role for CEP135 in angiogenesis in vivo.

Since CEP135 affected EC angiogenesis, its effects on the proliferation of HUVECs were assessed. Through CCK-8 and MTT assays, it was demonstrated that CEP135 knockdown markedly suppressed the proliferation and survival of ECs, as indicated by a decreased optical density value (Fig. 3A,B). These results indicated that CEP135 knockdown inhibited cell proliferation. Subsequently, the effects of CEP135 on cell cycle progression were analyzed by flow cytometry. Notably, CEP135 knockdown led to cell cycle arrest (Fig. 3C and Supplementary Fig. 3). In addition, the protein expression levels of CDK4 and cyclin D1, two markers of the cell cycle, were decreased following CEP135 knockdown in ECs (Fig. 3D–F; p 0.05). These findings indicated that CEP135 ablation suppressed HUVEC proliferation and cell cycle progression.

Fig. 3.

CEP135 knockdown suppresses HUVEC proliferation and cell cycle progression. (A) MTT and (B) Cell Counting Kit-8 assays revealed the effects of CEP135 on the proliferation of HUVECs posttransfection. (C) Flow cytometry showed the effect of CEP135 on the cell cycle progression of HUVECs posttransfection with the transfection of control and human CEP135 siRNA#1, and the percentage of cells at different stages of the cell cycle was analyzed. (D) Western blot analysis showed the effects of CEP135 on the expression levels of (E) CDK4 and (F) Cyclin D1 upon transfection with control and CEP135 siRNA#1. Each assay was performed 3 times. One-way ANOVA was used for hypothesis testing for significance in A and B. Error bars indicate SEM. ${}^{*}$p 0.05, ${}^{**}$p 0.01, ${}^{***}$p 0.001 vs. siControl.

CEP135 is a centrosomal protein that has the potential to affect mitosis [15]. Therefore, the present study measured the effects of CEP135 on cell division. Spindle orientation is important for many developmental processes, including cell division, epithelial tissue homeostasis and regeneration. Spindle orientation was determined by measuring the angle between the spindle axis and the substratum. Notably, knockdown of CEP135 resulted in an increase in spindle angle, indicative of defective spindle orientation, whereas spindle length was not affected (Fig. 4A–E). The model was shown in Fig. 4A. These findings suggested that CEP135 depletion led to defects in spindle orientation in ECs.

Fig. 4.

CEP135 knockdown affects the spindle orientation of HUVECs. (A) Scheme depicting spindle angle ($\alpha$) measurement. (B) Representative immunostaining images, (C) average spindle length, (D) average spindle angle and (E) percentage of cells with long astral microtubules. The position of the z stage is indicated in micrometers; 3D, xy projection. Cell numbers: N = 10 for each group. Error bars indicate SEM. Data were analyzed using unpaired Student’s t test. Scale bar, 5 µm. ${}^{**}$p 0.01, ${}^{***}$p 0.001 vs. siControl.

The present study then assessed the effect of CEP135 on HUVEC migration. A HUVEC monolayer was scratched with a pipette tip, and closure of the wounded area was documented to evaluate the capacity of cell migration. The wounded area in the control group was fully recovered after 24 h as a consequence of directed cell migration (Fig. 5A,B), suggesting the effects on cell migration. Transwell assays also revealed that CEP135 knockdown suppressed the invasion of HUVECs (Fig. 5C,D).

Fig. 5.

CEP135 is critical for vascular endothelial cell migration. (A) HUVECs transfected with siControl or siCEP135 were scratched, and images of the wound margins were captured after 0 and 24 h. (B) The extent of wound closure was semiquantified by measuring the wound area. (C) Cells invading the underside of the insert were stained with crystal violet. (D) Semiquantification of the number of invaded cells. Data were analyzed using one-way ANOVA. Error bars indicate SEM. Scale bar, 200 µm. ${}^{**}$p 0.01, ${}^{***}$p 0.001 vs. siControl. Each assay was performed 3 times. CEP135, centrosomal protein 135.

Polarization is a critical step in cell migration that involves the rearrangement of microtubules and reorientation of the centrosome [15]. HUVECs transfected with control or CEP135 siRNAs were scratched, and the cells were fixed 3 h later. In the control siRNA group, cells at the wound margin demonstrated a typical polarized structure, with the centrosome positioned between the nucleus and the leading edge (Fig. 6A,B). In contrast, the polarized morphology was suppressed in cells from the siCEP135 group (Fig. 6A). By semiquantifying the percentage of polarized cells, it was revealed that knockdown of CEP135 expression significantly inhibited cell polarization (Fig. 6C,D). In addition, the results indicated that CEP135 was critical for HUVEC migration and polarization by affecting the polarization angles (Fig. 6D).

Fig. 6.

CEP135 mediates vascular endothelial cell polarization. (A) Immunofluorescence microscopy images of siControl- and siCEP135-transfected HUVECs. $\alpha$-Tubulin (green) and $\gamma$-tubulin (red) staining showed polarization defects. The arrows indicate the polarization of HUVECs. (B) A model showing the measurement of the angle of polarization of cells. The (C) percentage and (D) angle of polarized cells were affected by siCEP135. Data were analyzed using one-way ANOVA. Error bars indicate SEM. Scale bar, 10 µm. Cell numbers: N = 10 for each group. ${}^{***}$p 0.001 vs. siControl.

The present study assessed the mechanism underlying the effects of CEP135 on EC proliferation and migration. The present study investigated whether CEP135 was involved in the regulation of microtubule stabilization, which is critical for cell polarization and migration. CEP135 knockdown significantly inhibited microtubule stabilization when cells were placed on ice for 30 min (Fig. 7A,B; p 0.05). To examine whether CEP135 directly regulates microtubule stability, tubulin polymerization was assessed using an in vitro tubulin turbidity assay. CEP135 was shown to increase tubulin turbidity (Fig. 7C) and protected the microtubules from dilution- and cooling-induced disassembly (Fig. 7D). Taken together, these findings indicated that CEP135 may influence microtubule stabilization via the regulation of microtubule rearrangement in ECs.

Fig. 7.

CEP135 is critical for vascular endothelial cell MT stabilization. (A) Immunofluorescence microscopy images of siControl- and siCEP135-transfected HUVECs. $\alpha$-Tubulin (green) and $\gamma$-tubulin (red) staining showed the MT stabilization of HUVECs on ice for 30 min. (B) Semiquantitative immunofluorescence. Cell numbers: N = 10 for each group. (C) Effects of purified CEP135 proteins (0, 10, 100 and 500 nM) on tubulin polymerization continuously measured for 40 min at a 350 nm wavelength at intervals of 30 sec. (D) MT depolymerization upon dilution and cooling was measured at a 350 nm wavelength. The arrow refers to the dilution time point. Data were analyzed using one-way ANOVA. Error bars indicate SEM. Scale bar, 50 µm. ${}^{**}$p 0.01, ${}^{***}$p 0.001 vs. siControl. Each assay was performed 3 times. CEP135, centrosomal protein 135; si, small interfering RNA; HUVECs, human umbilical vein endothelial cells; MT, microtubule.