University of Groningen
Klotho in vascular biology
Mencke, Rik
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Mencke, R. (2018). Klotho in vascular biology. Rijksuniversiteit Groningen.
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Chapter 9
Klotho deficiency promotes and induces the
development of intimal hyperplasia
R. Mencke M. Weij A. Smit-van Oosten A.T. Umbach J. Voelkl C. Leibrock G. Harms M. Bulthuis L. Quintanilla-Martinez F. Lang J.L. Hillebrands Manuscript in preparation
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Abstract
Klotho is a renal anti-ageing protein of which the circulating form has marked vascular effects. Klotho deficiency leads to vascular calcification, arteriolar hyalinosis, endothelial dysfunction, and arterial stiffening, whereas Klotho overexpression or supplementation has beneficial effects on the vasculature. It was noted in 1997 that Klotho deficiency did not only lead to vascular calcification, but also to intimal hyperplasia, but this finding has not been corroborated by subsequent studies.
To assess the relationship between Klotho and intima hyperplasia, we evaluated Klotho
-/-arteries and we used femoral artery endovascular wire injury and femoral artery cuffing as models of intimal hyperplasia in Klotho+/- and WT mice. We also evaluated arteries from kl/kl
mice (the original Klotho-deficient strain) and assessed the effect of spironolactone treatment. We furthermore studied the effect of Klotho on smooth muscle cell migration (SMC) in vitro.
We did not detect intimal hyperplasia in Klotho-/- arteries. Neointima formation induced by
wire injury was exacerbated in Klotho+/- mice compared to WT mice, while stenosis induced
by cuffing was increased as well. The latter, however, could be an effect of more severe thinning of the media in WT mice rather than a difference in neointima formation. Kl/kl mice did exhibit spontaneous intimal hyperplasia, which was more frequently found after spironolactone treatment. Finally, we found that recombinant Klotho directly inhibited SMC migration in vitro.
In conclusion, Klotho deficiency both induces and exacerbates the development of intima hyperplasia. The phenotypic variability seen in different Klotho-deficient mice and under different conditions hints at a central role for Klotho in regulating SMC differentiation, rendering Klotho potentially critical in preventing aberrant SMC behaviour in vascular diseases.
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Introduction
Klotho is a protein predominantly expressed in the kidney (1), where it functions as a co-receptor for fibroblast growth factor 23 (FGF23) (2, 3), and from where the soluble, circulating form of Klotho is derived (4). The premature ageing syndrome mice develop in Klotho deficiency (1) and the extension of lifespan by Klotho overexpression (5) have led to Klotho being considered an ageing suppressor. This phenotype of Klotho deficiency in mice includes a short lifespan, vascular calcification, osteoporosis, pulmonary emphysema, cognitive dysfunction, and hearing loss (1). The parallels to human ageing, a state of relative Klotho deficiency, and the premature ageing-like syndrome in end-stage renal disease (ESRD), a state of virtually complete Klotho deficiency (6-8), have long fuelled speculation that restoration of Klotho could be pivotal in the treatment of ageing-related and CKD-related diseases. Because Klotho protects against experimental renal disease (9-14), cardiac disease (10, 15-18), pulmonary disease (19, 20), neurodegenerative disease (21-24), and diabetes (25, 26), this approach is currently a very promising avenue for research.
The relationship between Klotho and the vasculature has been of particular interest. Since Klotho is principally expressed in the kidney, rather than in the vasculature (27), the vascular effects of Klotho are thought to be predominantly dependent on soluble Klotho in the circulation (4). Various degrees of Klotho deficiency are accompanied by various vascular abnormalities. It has long been known that full Klotho deficiency results in severe vascular calcification (1), which has been a major focus throughout the years. Less extensively established, however, are the facts that full Klotho deficiency can also lead to arteriolar hyalinosis and that partial Klotho deficiency causes endothelial dysfunction (28, 29), arterial stiffening (30, 31), and hypertension (32, 33). These vascular abnormalities play significant roles in the development of cardiovascular disease in ageing and in relation to chronic kidney disease (CKD), lending credence to a potential role for Klotho in combating cardiovascular disease. However, the original finding that Klotho-deficient mice display intimal hyperplasia (1) has not spurred any subsequent investigation into this phenomenon and it is currently unclear whether and under which conditions Klotho deficiency may result in intimal hyperplasia. Intimal hyperplasia is a common process in which medial smooth muscle cells (SMCs) de-differentiate from their contractile phenotype and migrate to the intima, where they proliferate and produce a narrowing of the vascular lumen, which occurs in atherosclerosis, in-stent re-stenosis after, e.g. percutaneous coronary intervention, and transplant vasculopathy. Because there are indications that Klotho can inhibit neointima and plaque formation (34-37), a potential role for Klotho in treating or preventing intimal hyperplasia merits further exploration. To address whether there is a relationship between Klotho and intimal hyperplasia, we investigated various Klotho-deficient mouse strains and we studied the development of intimal hyperplasia in femoral artery wire injury and femoral artery cuffing models of intimal hyperplasia.
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Materials and Methods
Animal experiments
Klotho+/- mice were kindly provided by Joost Hoenderop (Department of Physiology, Radboud
University, Nijmegen, The Netherlands) (38). Mice were bred at the Central Animal Facility of the University Medical Center Groningen, generating Klotho-/-, Klotho+/-, and WT mice. Mice
were housed with ad libitum access to chow and drinking water under 12-hour light/dark cycle conditions. At 8-12 weeks of age, Klotho+/- (N = 8) and WT mice (N = 8) (both male and female)
were anaesthetized using isoflurane anaesthesia and wire injury and cuffing were performed as described previously (39). Briefly, the skin was opened in the inguinal region, exposing the femoral artery. On one side, a 0.36 mm diameter angioplasty guide wire (Cook Medical Europe, Ltd., Ireland) was inserted into the lumen and was retracted three times. On the contralateral side, a non-constrictive polyethylene cuff (with an inner diameter of 0.4 mm, outer diameter of 0.8 mm, and a length of 2 mm; Portex, UK) was placed around the femoral artery. The skin was then sutured, analgesia (carprofen) was injected subcutaneously (5 mg/kg). Mice were housed alone for at least 24 hours after surgery. Animals were sacrificed by cardiac puncture and exsanguination under deep isoflurane anaesthesia. Three mice had died peroperatively. The experimental protocols for femoral wire injury and femoral cuffing were approved by the institutional ethical board and animal care and experimentation were performed in accordance with the NIH Guide for the Use and Care for Laboratory Animals.
Kl/kl mouse experiments were performed at the University of Tübingen and were not treated
(N = 15) or treated (N = 11) with spironolactone (80 mg/L in drinking water) between 3 and 8 weeks of age or with NH4Cl (0.28 M in drinking water).
Histochemical stainings
Arterial segments and kidneys were fixed in formalin and embedded in paraffin. Femoral artery blocks were cut with 3 µm sections being collected at 200 µm-spaced intervals for at least 10 intervals or until the tissue was fully cut. After de-paraffinizing and re-hydrating, Verhoeff stainings were performed by incubating sections with Verhoeff staining solution (5 parts 5% oxidized hematoxylin/ethanol, 2 parts 10% FeCl3, 2 parts 2% KI/1% I2), followed by
de-staining for 1 minute in 2% FeCl3 and Van Gieson counterstaining (14% thiazide/saturated
picric acid solution) for 3 minutes. Periodic acid-Schiff staining was performed by incubating sections in 1% periodic acid for 10 minutes and in Schiff’s reagent for 15 minutes followed by hematoxylin counterstaining. Hematoxylin-eosin stainings were performed by incubating sections for 10 minutes in hematoxylin, followed by rinsing in tap water, 2 minutes in eosin, and rinsing in ethanol.
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Morphometry
Slides were scanned using a Hamamatsu Nanozoomer 2.0HT (Hamamatsu Photonics, Hamamatsu, Japan) and morphometrical analyses were performed using Aperio ImageScope (Leica Biosystems, Germany). Intimal surface areas were determined by subtracting the surface area of the lumen from the surface area within the internal elastic lamina. The thickness of the media and intima was measured at four evenly spaced points for every artery. For kl/kl kidneys, the percentage of arteries in a section, ranging from the renal artery to interlobar arteries in size, with a discernible neointima was calculated.
Migration assays
Human aortic smooth muscle cells (HASMCs) (ScienCell, USA) were cultured in Smooth Muscle Cell Medium (SMCM, Lonza, USA), supplemented with 2% fetal calf serum (FCS), 1% penicillin/streptomycin, and 1% Smooth Muscle Growth Supplement (SMGS), all from Lonza. HASMCs were cultured at 37 °C and under 5% CO2 from passage 2-5. Transwell inserts with a
8.0 µm polycarbonate membrane for 24-well plates (Corning, USA) were coated with collagen type I (Sigma-Aldrich C3867) at a concentration of 1.5 mg/ml in 0.05% acetic acid/50% ethanol for 24 hours at 37 °C. Cells were serum-starved for 24 hours. A total of 650 µl of serum-free or 2% FCS-containing SMCM was added to the lower wells, before 20*103 cell were seeded
per insert in 150 µl serum-free SMCM. Recombinant human Klotho (R&D Systems, USA) was added to the cells in concentrations of 0.2 nM or 0.4 nM (if evenly diffused throughout the solution) or to the medium in the lower well as a chemotaxis control. After 24 hours, inserts were rinsed in PBS and fixed for 10 minutes in methanol at -20 °C, followed by rinsing with PBS and staining with hematoxylin for 10 minutes. Inserts were then rinsed with tap water, cells on the upper side of the membrane were thoroughly swiped off with a cotton swab, and the membranes were excised from the inserts, followed by mounting on glass slides in Kaiser’s glycerin with the outer side of the membrane upwards. Slides were scanned and cells in 5 fields of view were counted for each membrane. Experiments were performed three times independently.
Statistical analysis
Data are presented as mean ± standard deviation (SD) or as median [interquartile range] depending on the normality of the distribution. Normality was assessed using the Kolmogorov-Smirnov test. Differences between groups were tested using Student’s t test in case of a normal distribution or the Mann-Whitney U test in case of a non-normal distribution. Differences between more than two groups were compared using ANOVA followed by the Bonferroni post-hoc correction, or by Kruskal-Wallis followed by Dunn’s post-hoc correction,
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depending on the distribution. Statistical analyses were performed using GraphPad Prism software (version 5, GraphPad, USA). A p value < 0.05 was considered statistically significant.
Results
Evaluation of arteries in Klotho-/- mice
To assess whether we could confirm the findings originally reported by Kuro-o et al. detailing the presence of neointimas in intrarenal arteries in kl/kl kidneys (1), we investigated whether Klotho-/- mice develop spontaneous neointima formation in the aorta, in segmental renal
arteries, interlobular arteries, and arterioles. Although we detected arterial and arteriolar hyalinosis at the renal artery level and in arterioles, we did not detect intimal hyperplasia in the aorta (A), in segmental renal arteries (B), interlobular arteries (C), or arterioles (D) (Figure 1).
Figure 1. Evaluation of arteries in 7-weeks-old Klotho-/- mice. Hematoxylin-eosin stainings depicting (A) the aorta, (B) a renal segmental artery, (C) an interlobular artery, and (D) a renal arteriole reveal no intima
hyperplasia. Blue arrows indicate areas with accumulation of hyaline material and arteriolar hyalinosis. Green arrows indicate unaffected arteries/arterioles. Original magnifications are 400×.
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Wire injury-induced neointima formation in Klotho+/- mice
As the Klotho-/- mice used in this study developed a milder phenotype than expected in some
respects (notably with regard to vascular calcification), it was possible that a spontaneous neointima phenotype would similarly not overtly manifest. We therefore first decided to assess whether neointima formation induced by endovascular wire injury would be exaggerated in Klotho+/- mice, compared to WT littermates. At 4 weeks after injury, we found
that both Klotho+/- and WT mice had developed a neointima in the injured femoral artery
(Figure 2A, B). Klotho+/- mice had developed intimal lesions of on average, averaged over 9
cross-sections of the injured segment, 21836 ± 12165 µm², compared to 13512 ± 7171 µm² for WT mice (Figure 2C, p = 0.179). At the cross-section at which the lesions were the largest, Klotho+/- mice had developed significantly larger lesions (48850 ± 16179 µm²) than had WT
mice (31031 ± 10179 µm²) (Figure 2D, p < 0.05). In terms of relative occupancy of the lumen by (neo)intima, Klotho+/- lesions resulted in a stenosis with an average over the injured
segment of 56.2 ± 13.9%, compared to 38.9 ± 14.9% for WT lesions (Figure 2E), which almost reached statistical significance (p = 0.065). The maximal stenosis, which would be limiting in distal perfusion, was 82.3 ± 15.4% for Klotho+/- mice compared to 67.6 ± 18.1% for WT mice
(Figure 2F, p = 0.161). Although not all of these differences were statistically significant, overall it seems that neointima lesions in Klotho+/- induced by wire injury were larger and resulting in
a higher degree of stenosis, supporting the notion that Klotho deficiency exacerbates intimal hyperplasia when induced.
Cuffing-induced neointima formation in Klotho+/- mice
Having established that neointima formation in Klotho+/- mice is exacerbated in a severe
intimal hyperplasia model, we also opted for testing whether cuffing of the femoral artery, as a milder model of neointima formation with a different mechanism, would produce similar results. After 4 weeks, both Klotho+/- and WT mice had developed neointimas (Figure 3A, B),
although notably to a lesser extent compared to the contralateral (wire injury) side. Average Klotho+/- intimal surface areas were similar to WT lesions (5135 ± 3678 µm² vs 4423 ± 3840
µm², p = 0.738), as were the maximal intimal surface areas (10772 ± 6860 µm² vs 9781 ± 8868 µm², p = 0.824), depicted in Figure 3C, D. Klotho+/- mice, however, did develop a significantly
higher average degree of stenosis (22.4 ± 7.0%), compared to 14.1 ± 3.8% for WT mice (Figure 3E, p = 0.026). The maximal stenosis along the segment (Figure 3F) was 39.6 ± 20.7 for Klotho
+/-compared to 27.6 ± 13.7% for WT mice (p = 0.257). Taken together, it appears that a higher degree of luminal obstruction has developed in Klotho+/- mice, although not necessarily
because of a larger neointima. To address this finding and to investigate the differences between the models, we measured the thickness of the media and the intima for all arterial sections. After endovascular wire injury, the thickness of the media was similar across genotypes (Figure 4A), while the intima tended to be thicker in
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Figure 2. Morphometry on the neointima in femoral arteries in Klotho+/- and WT mice 4 weeks after wire injury.
(A) Representative Verhoeff staining from Klotho+/- femoral artery, featuring a neointima (arrow, denoted i). The internal elastic lamina is black and separates the intima from the media (denoted m). (B) Representative Verhoeff
staining from WT femoral artery, featuring a neointima (arrow). (C) Quantification of the intimal surface area,
averaged per mouse over 9 cross-sections, comparing Klotho+/- (N = 6) and WT mice (N = 6). (D) Quantification of the maximal intimal surface area in a segment per mouse, comparing Klotho+/- (N = 6) and WT (N = 6) mice. (E) Quantification of the luminal stenosis, averaged per mouse over 9 cross-sections, comparing Klotho+/- (N = 6) and WT mice (N = 6). (F) Quantification of the maximal stenosis in a segment per mouse, comparing Klotho+/- (N = 6) and WT (N = 6) mice. Data are presented as mean ± SD. * p < 0.05. Original magnifications are 300×.
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Figure 3. Morphometry on the neointima in femoral arteries in Klotho+/- and WT mice 4 weeks after cuffing.
(A) Representative Verhoeff staining from Klotho+/- femoral artery, featuring a neointima (arrow, denoted i). The internal elastic lamina is black and separates the intima from the media (denoted m). (B) Representative Verhoeff
staining from WT femoral artery, featuring a neointima (arrow). (C) Quantification of the intimal surface area,
averaged per mouse over 11 cross-sections, comparing Klotho+/- (N = 7) and WT mice (N = 6). (D) Quantification of the maximal intimal surface area in a segment per mouse, comparing Klotho+/- (N = 7) and WT (N = 6) mice. (E) Quantification of the luminal stenosis, averaged per mouse over 9 cross-sections, comparing Klotho+/- (N = 7) and WT mice (N = 6). (F) Quantification of the maximal stenosis in a segment per mouse, comparing Klotho+/- (N = 7) and WT (N = 6) mice. Data are presented as mean ± SD. * p < 0.05. Original magnifications are 300×.
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Klotho+/- mice (Figure 4B), corresponding with the larger surface area in Figure 2C, D and
resulting in a higher intima-media thickness (IMT) ratio (Figure 4C), albeit not significantly. After femoral cuffing, the average thickness of the media decreased in WT mice (p = 0.089, Figure 4D, also note the thin media in Figure 3B) while the average thickness of the intima was similar (Figure 4E) and the IMT ratio was higher in WT mice, although these differences were not significant. Speculatively, it appears that overall, cuffing in Klotho+/- mice may have resulted in a higher degree of stenosis mostly due to a lack of compensatory thinning of the media with a comparative widening of the vessel lumen. Iliac artery segments proximal to the injured femoral segments were considered sham segments and were unaffected (Supplemental Figure 1).
Evaluation of arteries in kl/kl mice
Having assessed Klotho-/- mouse arteries, we then investigated whether spontaneous intimal
hyperplasia develops in the original kl/kl mouse (which has a disrupted Klotho promoter rather than a deleted exon and which also has a different genetic background). We found that neointima formation sporadically occurs in kl/kl mice, most notably in (segmental) renal
Figure 4. Morphometry on the vascular wall layers in femoral arteries from Klotho+/- and WT mice 4 weeks
after wire injury and cuffing. (A) Average thickness of the media (averaged over the segment and measured at 4
evenly spaced points for every artery), comparing Klotho+/- and WT mice after wire injury. (B) Average thickness of the intima, comparing Klotho+/- and WT mice after wire injury. (C) Average intima-media thickness (IMT) ratio, comparing Klotho+/- and WT mice after wire injury. (D) Average thickness of the media, comparing Klotho+/- and WT mice after femoral cuffing. (E) Average thickness of the intima, comparing Klotho+/- and WT mice after femoral cuffing. (F) Average intima-media thickness (IMT) ratio, comparing Klotho+/- and WT mice after femoral cuffing. Data are presented as mean ± SD.
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arteries (Figure 5A, C). Kl/kl arteries, however, are predominantly affected by severe vascular calcification. We have previously shown that inhibition of vascular calcification by spironolactone (as described in (40)) potentiated the development of hyalinosis. We similarly found that treatment of kl/kl mice with spironolactone potentiated the development of intimal hyperplasia (Figure 5B,D), which was significantly more common in arteries (p = 0.004, Figure 5E). To underscore the point that this is not a spironolactone-specific effect, but rather appears to be a function of the degree to which vascular calcification is inhibited, we also detected marked neointima formation in kl/kl mice treated with NH4Cl (Supplemental Figure
2), which has previously been shown to inhibit vascular calcification in kl/kl mice almost completely ((41).
Klotho inhibits migration of smooth muscle cells
The combination of spontaneously developing intimal hyperplasia in Klotho deficiency raises the question whether soluble Klotho may directly influence SMC migration. We seeded human aortic SMCs in Boyden chamber inserts, treated with 0, 0.2, or 0.4 nM of recombinant Klotho, and investigated spontaneous migration to the lower well (in a 0% FCS
Figure 5. Evaluation of neointima formation in untreated and spironolactone-treated kl/kl mice. (A) Periodic
acid-Schiff (PAS) staining showing spontaneous intimal hyperplasia in an untreated kl/kl mouse renal artery. Original magnification is 160×. (B) PAS staining showing marked spontaneous intimal hyperplasia in a
spironolactone-treated kl/kl mouse renal artery. Original magnification is 160×. (C) Inset from (A). Original
magnification is 400×. (D) Inset from (B). Original magnification is 160×. (E) Quantification of the occurrence of
intimal hyperplasia in untreated (N = 15) and spironolactone-treated (N = 11) kl/kl mice, expressed as the percentage of arteries (between the renal artery and interlobar arteries) with intimal hyperplasia. Data are presented as mean ± SD. ** p < 0.01. Arrows indicate neointimas.
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condition) or serum-stimulated migration to the lower well (in a 2% FCS condition). We found that Klotho dose-dependently inhibited unstimulated SMC migration (Figure 6A, B; p < 0.05 for 0.4 nM vs 0 nM Klotho) by about 35%. SMC migration stimulated by 2% FCS was similarly inhibited by Klotho (Figure 6C, D), but these difference did not reach statistical significance. As a control, we assessed whether Klotho would have a chemoattractant effect on SMCs by adding Klotho in the lower chamber. We observed a modest but significant decrease in SMC migration of about 10% by a recombinant Klotho gradient diffusing from the lower well side (Figure 6E, F). These results indicate that Klotho directly inhibits migration of SMCs.
Figure 6. Recombinant Klotho inhibits smooth muscle cell (SMC) migration in vitro. (A) Quantification of average
cell numbers standardized to the control condition. Klotho dose-dependently decreased unstimulated SMC migration. (B) Hematoxylin staining of migrated SMCs with SMC culture medium with 0% fetal calf serum (FCS)
in the lower well, after stimulation with 0, 0.2, or 0.4 nM of recombinant Klotho for 24 hours. (C) Quantification
of average cell numbers standardized to the control condition, indicating SMC migration stimulated by 2% FCS in the lower well. (D) Hematoxylin staining of migrated SMCs with SMC culture medium with 2% fetal calf serum
(FCS) in the lower well, after stimulation with 0, 0.2, or 0.4 nM of recombinant Klotho for 24 hours. (E)
Quantification of average cell numbers standardized to the control condition, indicating that Klotho does not act as a chemo-attractant and in a gradient from the lower well decreases SMC migration. (F) Hematoxylin staining
of migrated SMCs with SMC culture medium with 0% fetal calf serum (FCS) and 0 or 0.4 nM Klotho in the lower well, for 24 hours. Each data point indicates an average of 5 fields of view and each experiment was performed three times independently. Data are presented as mean ± SD. * p < 0.05.
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Discussion
This study is the first evaluation of the relationship between intimal hyperplasia and Klotho deficiency since spontaneous neointima formation was noted in the original paper in which Klothowasfirst described (1). We found that partial Klotho deficiency in Klotho+/- mice
exacerbates intimal hyperplasia induced by endovascular femoral artery wire injury as well as femoral artery cuffing-induced stenosis. We further confirmed the finding that neointima formation occurs spontaneously in kl/kl mice (but not in Klotho-/- mice) and that this process
is potentiated by pharmacological inhibition of vascular calcification development. Finally, the role for Klotho in the process of intimal hyperplasia may include direct inhibitions of SMC migration.
A number of possibilities could account for the differential response of Klotho+/- mice to the
two different intimal hyperplasia models employed in this study. First of all, it is possible that the cuffing model was too mild to allow for a difference to materialize, compared to the more severe wire injury model with more pronounced neointima formation and more pronounced differences between Klotho+/- and WT mice. Secondly, the mechanism of induction of intimal
hyperplasia is different between the two models, suggesting that it could be the case that partial Klotho deficiency has differential effects in the sense that the result of primary adventitial injury (42) could be less modifiable by Klotho than primary endovascular endothelial denudation. The tendency for the media to become thinner in response to the presence of a cuff in WT mice compared to Klotho+/- mice could be suggestive of a
compensatory mechanism to prevent stenosis of the lumen, while at the same time predisposing to aneurysm formation, which would not be beneficial. Replication experiments and additional studies, including Klotho treatment of WT mice or Klotho overexpression, are required to determine whether this represents an actual effect and whether it would represent an effect in WT mice resulting from the presence of more Klotho, the lack of dysregulation in a multitude of pathways down-stream of Klotho deficiency affecting the media, and whether the neointima responses in Klotho+/- and WT mice at a later time point
are still keeping pace or may result in a more (clinically) relevant degree of luminal obstruction or aneurysm development. Finally, we should be mindful that the large variation in both cuffing and wire injury models leaves us underpowered and the data are currently inconclusive.
As we found previously, there exists a degree of phenotypic variability when it comes to different Klotho-deficient mouse strains, for which the genetic background, diet, and method of precluding Klotho protein translation appear to be responsible. We have previously shown that kl/kl mice predominantly exhibit marked vascular calcification and Klotho-/- mice are
mostly affected by arteriolar hyalinosis, while arteriolar hyalinosis also occurs in kl/kl mice when the development of vascular calcification is inhibited. While the aetiology of these
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vascular phenotypes is different, one commonality between them is the emergent aberrant behaviour of de-differentiated SMCs. These SMCs can undergo a phenotypic shift from their normal, contractile phenotype, to a calcifying phenotype with traits of osteochondrogenic cells, or to a synthetic phenotype. Confirmation of the finding that Klotho deficiency can spontaneously lead to neointima formation allows us to speculate about the role of Klotho in this process, in which SMCs de-differentiate and assume a more migratory and proliferative phenotype. Combined with the established effects of Klotho in the development of vascular calcification and arteriolar hyalinosis, the findings that Klotho deficiency influences neointima formation in different Klotho-deficient mouse strains and even allows for the spontaneous development of intimal hyperplasia as part of the phenotype of Klotho deficiency (albeit to a varying degree) is of particular interest. We hypothesize that Klotho may principally affect SMC differentiation and that loss of Klotho predisposes SMCs to de-differentiation, while assuming any of a host of different, aberrant SMC phenotypes, depending on the stimuli involved. Indeed, it has been shown by Hu et al. that stimulation of SMCs with recombinant Klotho promotes differentiation towards a contractile phenotype, including an increased expression of SM22α and α-SMA, with an accompanying decrease in expression of osteochondrogenic genes, which is in support of this hypothesis (7). The observation that inhibition of vascular calcification to an extent results in the consequent emergence of a number of different vascular lesions (in which SMCs still play pathological roles) also offers a new perspective on the implied general view perpetuated in vascular calcification studies in Klotho knockout mice, which is that if vascular calcification is inhibited by whatever means, the arteries subsequently normalize. However, our data indicate that a lack of Klotho still renders the vasculature prone to developing various other lesions in lieu of calcification. The wide array of vascular abnormalities observed in Klotho deficiency, which in human disease are generally not considered to share a common aetiology, also endorses the notion that Klotho deficiency could be a factor highly up-stream mechanistically and a primary problem in itself for the vasculature (including endothelial and smooth muscle cells). The fact that all of the vascular abnormalities observed in Klotho deficiency are also lesions that occur in human ageing underscores the potential relevance for Klotho in influencing ageing-related vascular disease.
The potential of a Klotho-based treatment to prevent neointima formation was recently explored in a number of ways. Firstly, Kamari et al. did not detect a difference in aortic plaque formation in ApoE-/- mice, which of course is a model in which lipid metabolism is an important
aetiological factor as well, in contrast to the models used in this study (43). However, in the study by Kaminari et al., there was also no effect of Klotho on blood pressure (which would have been expected (44, 45)) and a later study by Yang et al. did identify a clear effect of Klotho treatment on plaque formation in both ApoE-/- mice with CKD or treated with indoxyl sulphate
(IS) (34). It is possible, however, that suppression of Klotho expression in experimental of CKD or by IS treatment (46) and subsequent restoration of Klotho levels results in a beneficial effect, while additional Klotho supplementation in ApoE-/- mice is ineffective. Varshney et al.
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and pulmonary artery remodelling and intima formation (47). Future studies are necessary in order to provide conclusive answers. Mechanistically more similar to the wire injury (endothelial denudation) model used in this study is a number of rat experiments performed by Chen et al., who transplanted de-cellularized rat carotid arteries as tissue-engineered blood vessels (TBEVs) and found marked inhibitory effects of Klotho-containing nanoparticles (36) and of Klotho- (and ADK siRNA-)containing exosomes (37) on the rate of occlusion and the development of intimal hyperplasia.
In conclusion, this is the first study to confirm that neointima formation occurs spontaneously in and is exacerbated by Klotho deficiency, possibly due to an inhibitory effect of Klotho on SMC migratory capacity. We hypothesize that loss of Klotho is critical in SMC de-differentiation and an array of associated vascular diseases. Future studies will be required to elucidate the molecular mechanism responsible for Klotho-SMC interactions and to explore whether there could be a therapeutic role for Klotho in treating vascular conditions characterized by intimal hyperplasia.
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Acknowledgments
We are grateful for the contributions of Bianca Schepers-Meijeringh, Erjan Hilbrands, Marjan Reinders-Luinge, and Monique Lodewijk. This study was funded by a consortium grant of the Dutch Kidney Foundation [CP10.11] (NIGRAM PIs: Joost Hoenderop and René Bindels (Radboud University Medical Center, Nijmegen), Piet ter Wee and Marc Vervloet (VU University Medical Center, Amsterdam), and Gerjan Navis, Martin de Borst, and Jan-Luuk Hillebrands (University Medical Center Groningen). This study was also funded by the University Medical Center Groningen MD/PhD program.
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References
1. M. Kuro-o, Y. Matsumura, H. Aizawa, H. Kawaguchi, T. Suga, T. Utsugi, Y. Ohyama, M. Kurabayashi, T. Kaname, E. Kume, H. Iwasaki, A. Iida, T. Shiraki-Iida, S. Nishikawa, R. Nagai, Y. I. Nabeshima, Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature. 390, 45-51 (1997).
2. H. Kurosu, Y. Ogawa, M. Miyoshi, M. Yamamoto, A. Nandi, K. P. Rosenblatt, M. G. Baum, S. Schiavi, M. C. Hu, O. W. Moe, M. Kuro-o, Regulation of fibroblast growth factor-23 signaling by klotho. J. Biol. Chem. 281,
6120-6123 (2006).
3. I. Urakawa, Y. Yamazaki, T. Shimada, K. Iijima, H. Hasegawa, K. Okawa, T. Fujita, S. Fukumoto, T. Yamashita, Klotho converts canonical FGF receptor into a specific receptor for FGF23. Nature. 444, 770-774 (2006).
4. K. Lindberg, R. Amin, O. W. Moe, M. C. Hu, R. G. Erben, A. Ostman Wernerson, B. Lanske, H. Olauson, T. E. Larsson, The kidney is the principal organ mediating klotho effects. J. Am. Soc. Nephrol. 25, 2169-2175 (2014).
5. H. Kurosu, M. Yamamoto, J. D. Clark, J. V. Pastor, A. Nandi, P. Gurnani, O. P. McGuinness, H. Chikuda, M. Yamaguchi, H. Kawaguchi, I. Shimomura, Y. Takayama, J. Herz, C. R. Kahn, K. P. Rosenblatt, M. Kuro-o, Suppression of aging in mice by the hormone Klotho. Science. 309, 1829-1833 (2005).
6. N. Koh, T. Fujimori, S. Nishiguchi, A. Tamori, S. Shiomi, T. Nakatani, K. Sugimura, T. Kishimoto, S. Kinoshita, T. Kuroki, Y. Nabeshima, Severely reduced production of klotho in human chronic renal failure kidney. Biochem.
Biophys. Res. Commun. 280, 1015-1020 (2001).
7. M. C. Hu, M. Shi, J. Zhang, H. Quinones, C. Griffith, M. Kuro-o, O. W. Moe, Klotho deficiency causes vascular calcification in chronic kidney disease. J. Am. Soc. Nephrol. 22, 124-136 (2011).
8. S. L. Barker, J. Pastor, D. Carranza, H. Quinones, C. Griffith, R. Goetz, M. Mohammadi, J. Ye, J. Zhang, M. C. Hu, M. Kuro-o, O. W. Moe, S. S. Sidhu, The demonstration of alphaKlotho deficiency in human chronic kidney disease with a novel synthetic antibody. Nephrol. Dial. Transplant. 30, 223-233 (2015).
9. M. C. Hu, M. Shi, J. Zhang, H. Quinones, M. Kuro-o, O. W. Moe, Klotho deficiency is an early biomarker of renal ischemia-reperfusion injury and its replacement is protective. Kidney Int. 78, 1240-1251 (2010).
10. M. C. Hu, M. Shi, N. Gillings, B. Flores, M. Takahashi, M. Kuro-O, O. W. Moe, Recombinant alpha-Klotho may be prophylactic and therapeutic for acute to chronic kidney disease progression and uremic cardiomyopathy.
Kidney Int. (2017).
11. X. Guan, L. Nie, T. He, K. Yang, T. Xiao, S. Wang, Y. Huang, J. Zhang, J. Wang, K. Sharma, Y. Liu, J. Zhao, Klotho suppresses renal tubulo-interstitial fibrosis by controlling basic fibroblast growth factor-2 signalling. J. Pathol.
234, 560-572 (2014).
12. S. Doi, Y. Zou, O. Togao, J. V. Pastor, G. B. John, L. Wang, K. Shiizaki, R. Gotschall, S. Schiavi, N. Yorioka, M. Takahashi, D. A. Boothman, M. Kuro-o, Klotho inhibits transforming growth factor-beta1 (TGF-beta1) signaling and suppresses renal fibrosis and cancer metastasis in mice. J. Biol. Chem. 286, 8655-8665 (2011).
302
13. M. C. Panesso, M. Shi, H. J. Cho, J. Paek, J. Ye, O. W. Moe, M. C. Hu, Klotho has dual protective effects on cisplatin-induced acute kidney injury. Kidney Int. 85, 855-870 (2014).
14. M. Shi, B. Flores, N. Gillings, A. Bian, H. J. Cho, S. Yan, Y. Liu, B. Levine, O. W. Moe, M. C. Hu, alphaKlotho Mitigates Progression of AKI to CKD through Activation of Autophagy. J. Am. Soc. Nephrol. (2015).
15. M. C. Hu, M. Shi, H. J. Cho, B. Adams-Huet, J. Paek, K. Hill, J. Shelton, A. P. Amaral, C. Faul, M. Taniguchi, M. Wolf, M. Brand, M. Takahashi, M. Kuro-O, J. A. Hill, O. W. Moe, Klotho and Phosphate Are Modulators of Pathologic Uremic Cardiac Remodeling. J. Am. Soc. Nephrol. (2014).
16. J. Xie, S. K. Cha, S. W. An, M. Kuro-O, L. Birnbaumer, C. L. Huang, Cardioprotection by Klotho through downregulation of TRPC6 channels in the mouse heart. Nat. Commun. 3, 1238 (2012).
17. J. Xie, J. Yoon, S. W. An, M. Kuro-O, C. L. Huang, Soluble Klotho Protects against Uremic Cardiomyopathy Independently of Fibroblast Growth Factor 23 and Phosphate. J. Am. Soc. Nephrol. (2014).
18. K. Yang, C. Wang, L. Nie, X. Zhao, J. Gu, X. Guan, S. Wang, T. Xiao, X. Xu, T. He, X. Xia, J. Wang, J. Zhao, Klotho Protects Against Indoxyl Sulphate-Induced Myocardial Hypertrophy. J. Am. Soc. Nephrol. (2015).
19. P. Ravikumar, L. Li, J. Ye, M. Shi, M. Taniguchi, J. Zhang, M. Kuro-O, M. C. Hu, O. W. Moe, C. C. Hsia, Alpha-Klotho deficiency in Acute Kidney Injury Contributes to Lung Damage. J. Appl. Physiol. (1985)., jap.00792.2015 (2015).
20. P. Ravikumar, J. Ye, J. Zhang, S. N. Pinch, M. C. Hu, M. Kuro-o, C. C. Hsia, O. W. Moe, alpha-Klotho protects against oxidative damage in pulmonary epithelia. Am. J. Physiol. Lung Cell. Mol. Physiol. 307, L566-75 (2014).
21. D. B. Dubal, L. Zhu, P. E. Sanchez, K. Worden, L. Broestl, E. Johnson, K. Ho, G. Q. Yu, D. Kim, A. Betourne, M. Kuro-O, E. Masliah, C. R. Abraham, L. Mucke, Life extension factor klotho prevents mortality and enhances cognition in hAPP transgenic mice. J. Neurosci. 35, 2358-2371 (2015).
22. D. B. Dubal, J. S. Yokoyama, L. Zhu, L. Broestl, K. Worden, D. Wang, V. E. Sturm, D. Kim, E. Klein, G. Q. Yu, K. Ho, K. E. Eilertson, L. Yu, M. Kuro-o, P. L. De Jager, G. Coppola, G. W. Small, D. A. Bennett, J. H. Kramer, C. R. Abraham, B. L. Miller, L. Mucke, Life extension factor klotho enhances cognition. Cell. Rep. 7, 1065-1076 (2014).
23. A. Masso, A. Sanchez, L. Gimenez-Llort, J. M. Lizcano, M. Canete, B. Garcia, V. Torres-Lista, M. Puig, A. Bosch, M. Chillon, Secreted and Transmembrane alphaKlotho Isoforms Have Different Spatio-Temporal Profiles in the Brain during Aging and Alzheimer's Disease Progression. PLoS One. 10, e0143623 (2015).
24. J. Leon, A. J. Moreno, B. I. Garay, R. J. Chalkley, A. L. Burlingame, D. Wang, D. B. Dubal, Peripheral Elevation of a Klotho Fragment Enhances Brain Function and Resilience in Young, Aging, and alpha-Synuclein Transgenic Mice. Cell. Rep. 20, 1360-1371 (2017).
25. Y. Lin, Z. Sun, In Vivo Pancreatic beta-Cell-Specific Expression of Antiaging Gene Klotho: A Novel Approach for Preserving beta-Cells in Type 2 Diabetes. Diabetes. 64, 1444-1458 (2015).
26. Y. Lin, Z. Sun, Anti-aging Gene Klotho Attenuates Pancreatic beta Cell Apoptosis in Type I Diabetes. Diabetes. (2015).
303
27. R. Mencke, G. Harms, K. Mirkovic, J. Struik, J. Van Ark, E. Van Loon, M. Verkaik, M. H. De Borst, C. J. Zeebregts, J. G. Hoenderop, M. G. Vervloet, J. L. Hillebrands, NIGRAM Consortium, NIGRAM consortium, Membrane-bound Klotho is not expressed endogenously in healthy or uraemic human vascular tissue. Cardiovasc. Res. (2015). 28. T. Nakamura, Y. Saito, Y. Ohyama, H. Masuda, H. Sumino, M. Kuro-o, Y. Nabeshima, R. Nagai, M. Kurabayashi, Production of nitric oxide, but not prostacyclin, is reduced in klotho mice. Jpn. J. Pharmacol. 89, 149-156 (2002).
29. Y. Saito, T. Yamagishi, T. Nakamura, Y. Ohyama, H. Aizawa, T. Suga, Y. Matsumura, H. Masuda, M. Kurabayashi, M. Kuro-o, Y. Nabeshima, R. Nagai, Klotho protein protects against endothelial dysfunction. Biochem. Biophys.
Res. Commun. 248, 324-329 (1998).
30. D. Gao, Z. Zuo, J. Tian, Q. Ali, Y. Lin, H. Lei, Z. Sun, Activation of SIRT1 Attenuates Klotho Deficiency-Induced Arterial Stiffness and Hypertension by Enhancing AMP-Activated Protein Kinase Activity. Hypertension. 68,
1191-1199 (2016).
31. K. Chen, X. Zhou, Z. Sun, Haplodeficiency of Klotho Gene Causes Arterial Stiffening via Upregulation of Scleraxis Expression and Induction of Autophagy. Hypertension. 66, 1006-1013 (2015).
32. X. Zhou, K. Chen, H. Lei, Z. Sun, Klotho gene deficiency causes salt-sensitive hypertension via monocyte chemotactic protein-1/CC chemokine receptor 2-mediated inflammation. J. Am. Soc. Nephrol. 26, 121-132
(2015).
33. X. Zhou, K. Chen, Y. Wang, M. Schuman, H. Lei, Z. Sun, Antiaging Gene Klotho Regulates Adrenal CYP11B2 Expression and Aldosterone Synthesis. J. Am. Soc. Nephrol. (2015).
34. K. Yang, C. Du, X. Wang, F. Li, Y. Xu, S. Wang, S. Chen, F. Chen, M. Shen, M. Chen, M. Hu, T. He, Y. Su, J. Wang, J. Zhao, Uremic solute indoxyl sulfate-induced platelet hyperactivity contributes to CKD-associated thrombosis in mice. Blood. (2017).
35. L. Shu, M. Chen, M. Wen, C. Huang, J. Xu, Study of klotho gene transfer for the protective effect of the coronary of diabetic rats. Pak. J. Pharm. Sci. 27, 2095-2099 (2014).
36. W. Chen, F. Wang, W. Zeng, J. Sun, L. Li, M. Yang, J. Sun, Y. Wu, X. Zhao, C. Zhu, Regulation of Cellular Response Pattern to Phosphorus Ion is a New Target for the Design of Tissue-Engineered Blood Vessel. Adv. Healthc. Mater. (2015).
37. W. Chen, M. Yang, J. Bai, X. Li, X. Kong, Y. Gao, L. Bi, L. Xiao, B. Shi, Exosome-Modified Tissue Engineered Blood Vessel for Endothelial Progenitor Cell Capture and Targeted siRNA Delivery. Macromol. Biosci. (2017). 38. T. E. Woudenberg-Vrenken, B. C. van der Eerden, A. W. van der Kemp, J. P. van Leeuwen, R. J. Bindels, J. G. Hoenderop, Characterization of vitamin D-deficient klotho(-/-) mice: do increased levels of serum 1,25(OH)2D3 cause disturbed calcium and phosphate homeostasis in klotho(-/-) mice? Nephrol. Dial. Transplant. 27,
4061-4068 (2012).
39. J. Moser, J. van Ark, M. C. van Dijk, D. L. Greiner, L. D. Shultz, H. van Goor, J. L. Hillebrands, Distinct Differences on Neointima Formation in Immunodeficient and Humanized Mice after Carotid or Femoral Arterial Injury. Sci.
304
40. J. Voelkl, I. Alesutan, C. B. Leibrock, L. Quintanilla-Martinez, V. Kuhn, M. Feger, S. Mia, M. S. Ahmed, K. P. Rosenblatt, M. Kuro-O, F. Lang, Spironolactone ameliorates PIT1-dependent vascular osteoinduction in klotho-hypomorphic mice. J. Clin. Invest. 123, 812-822 (2013).
41. C. B. Leibrock, I. Alesutan, J. Voelkl, T. Pakladok, D. Michael, E. Schleicher, Z. Kamyabi-Moghaddam, L. Quintanilla-Martinez, M. Kuro-O, F. Lang, NH4Cl Treatment Prevents Tissue Calcification in Klotho Deficiency. J.
Am. Soc. Nephrol. (2015).
42. J. Wang, Y. Wang, J. Wang, X. Guo, E. C. Chan, F. Jiang, Adventitial Activation in the Pathogenesis of Injury-Induced Arterial Remodeling: Potential Implications in Transplant Vasculopathy. Am. J. Pathol. (2018). 43. Y. Kamari, O. Fingrut, A. Shaish, T. Almog, M. Kandel-Kfir, D. Harats, T. Rubinek, I. Wolf, The Effect of Klotho Treatment on Atherogenesis, Blood Pressure, and Metabolic Parameters in Experimental Rodent Models. Horm.
Metab. Res. (2015).
44. L. Zhou, H. Mo, J. Miao, D. Zhou, R. J. Tan, F. F. Hou, Y. Liu, Klotho Ameliorates Kidney Injury and Fibrosis and Normalizes Blood Pressure by Targeting the Renin-Angiotensin System. Am. J. Pathol. 185, 3211-3223 (2015).
45. Y. Wang, Z. Sun, Klotho gene delivery prevents the progression of spontaneous hypertension and renal damage. Hypertension. 54, 810-817 (2009).
46. C. Y. Sun, S. C. Chang, M. S. Wu, Suppression of Klotho expression by protein-bound uremic toxins is associated with increased DNA methyltransferase expression and DNA hypermethylation. Kidney Int. 81,
640-650 (2012).
47. R. Varshney, Q. Ali, C. Wu, Z. Sun, Monocrotaline-Induced Pulmonary Hypertension Involves Downregulation of Antiaging Protein Klotho and eNOS Activity. Hypertension. 68, 1255-1263 (2016).
305
Supplemental Figure 1. Verhoeff stainings on sham iliac arteries in Klotho+/- and WT mice 4 weeks after distal
wire injury or cuffing. (A) Representative Verhoeff staining from a normal Klotho+/- iliac artery proximal to femoral wire injury. (B) Representative Verhoeff staining from a normal WT iliac artery proximal to femoral wire
injury. (C) Representative Verhoeff staining from a normal Klotho+/- iliac artery, proximal to femoral cuffing. (D) Representative Verhoeff staining from a normal WT iliac artery, proximal to femoral cuffing. Original magnifications are 400×.
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Supplemental Figure 2. Evaluation of neointima formation in NH4Cl-treated kl/kl mice. (A) Periodic acid-Schiff (PAS) staining showing spontaneous intimal hyperplasia in a NH4Cl-treated kl/kl mouse renal artery. Original magnification is 160×. (B) Inset from (A). Original magnification is 400×. Arrows indicate neointima.
