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. 2025 Jan 3;378(1):1–9. doi: 10.1124/jpet.120.000424

The Caveolin-1 Scaffolding Domain Peptide Reverses Aging-Associated Deleterious Changes in Multiple Organs

Dhandapani Kuppuswamy 1,*, Panneerselvam Chinnakkannu 1, Charles Reese 1, Stanley Hoffman 1
PMCID: PMC13102142  PMID: 33879542

Abstract

Aging is a progressive, multifactorial, degenerative process in which deleterious changes occur in the biochemistry and function of organs. We showed that angiotensin II (AngII)-induced pathologies in the heart and kidney of young (3-month-old) mice are suppressed by the caveolin-1 scaffolding domain (CSD) peptide. Because AngII mediates many aging-associated changes, we explored whether CSD could reverse pre-existing pathologies and improve organ function in aged mice. Using 18-month-old mice (similar to 60-year-old humans), we found that >5-fold increases in leakage of serum proteins and >2-fold increases in fibrosis are associated with aging in the heart, kidney, and brain. Because tyrosine phosphorylation of cell junction proteins leads to the loss of microvascular barrier function, we analyzed the activation of the receptor tyrosine kinase PDGFR and the nonreceptor tyrosine kinases c-Src and Pyk2. We observed 5-fold activation of PDGFR and 2- to 3-fold activation of c-Src and Pyk2 in aged mice. Treatment with CSD for 4 weeks reversed these pathologic changes (microvascular leakage, fibrosis, kinase activation) in all organs almost down to the levels in healthy, young mice. In studies of heart function, CSD reduced the aging-associated increase in cardiomyocyte cross-sectional area and enhanced ventricular compliance in that echocardiographic studies demonstrated improved ejection fraction and fractional shortening and reduced isovolumic relation time. These results suggest that versions of CSD may be developed as treatments for aging-associated diseases in human patients based on the concept that CSD inhibits tyrosine kinases, leading to the inhibition of microvascular leakage and associated fibrosis, thereby improving organ function.

SIGNIFICANCE STATEMENT

The caveolin-1 scaffolding domain (CSD) peptide reverses aging-associated fibrosis, microvascular leakage, and organ dysfunction in the heart, kidneys, and brain via a mechanism that involves the suppression of the activity of multiple tyrosine kinases, suggesting that CSD can be developed as a treatment for a wide range of diseases found primarily in the aged.

Introduction

Aging contributes to organ failure in multiple tissues, including heart, kidney, and brain (Gurtner et al., 2008; Laflamme and Murry, 2011; Singh and Newman, 2011; Hecker et al., 2014). For example, heart function is progressively compromised in terms of diastolic and systolic compliance, kidneys lose the ability to filter waste products, and changes in the brain increase the risk for dementia. The immediate insults to the tissues that cause the effects of aging include excessive extracellular matrix deposition (fibrosis) and microvascular leakage (Weis, 2008; Boyle et al., 2011; Rosenberg, 2014; Saez and Vilchez, 2014; Piek et al., 2016; Toba and Lindsey, 2019). Fibrosis and microvascular leakage also occur in disease models for fibrosis in young animals treated with bleomycin (primarily targeting lung and skin) (Kasper et al., 1998; Tourkina et al., 2005; Del Galdo et al., 2008; Tourkina et al., 2010, 2011; Lee et al., 2014a,b, 2015; Reese et al., 2014a) or with angiotensin II (AngII) (primarily targeting heart and kidney) (Chinnakkannu et al., 2018).

Microvascular leakage is observed in a variety of devastating diseases associated with aging, including lung fibrosis, kidney fibrosis, congestive heart failure, and neurodegenerative diseases (Lakatta and Levy, 2003; Brandes et al., 2005; Oakley and Tharakan, 2014). Increases in ROS are a major cause of leakage because ROS activate receptor and nonreceptor tyrosine kinases, resulting in the phosphorylation of several cell junctional proteins, which allows the entry of both serum proteins and inflammatory cells into tissues (i.e., inflammation) (Reddy et al., 1995; Garrido and Griendling, 2009; Foulquier et al., 2018). In the aging heart, this enhanced inflammation leads to reactive myocardial fibrosis, which contributes to maladaptive ventricular remodeling, myocyte hypertrophy, and cardiac dysfunction, including loss of systolic and diastolic compliance (Piek et al., 2016). In summary, microvascular leakage, inflammation, and interstitial fibrosis are intertwined processes that have a pathologic effect on organ function.

Caveolin-1 is a structural cytoplasmic membrane protein involved in the formation/function of caveolae (Williams and Lisanti, 2004). Besides its role in endocytosis and receptor trafficking, caveolin-1 is a master regulatory protein that inhibits a wide range of kinases, including nonreceptor and receptor tyrosine kinases, by binding to these proteins via the caveolin-1 scaffolding domain (CSD, amino acids 82-101 of caveolin-1) (Couet et al., 1997; Oka et al., 1997; Rybin et al., 1999; Razani et al., 2001; Tourkina et al., 2005; Le Saux et al., 2008; Wang et al., 2008; Bhandary et al., 2012). CSD reverses the effects of low caveolin-1 on fibroblasts (Col I overproduction), monocytes (enhanced migration in vitro/recruitment in vivo and differentiation into fibroblastic cells), and mesenchymal stem cells (Col I overproduction) from patients with scleroderma and bleomycin-treated mice (Kasper et al., 1998; Tourkina et al., 2005, 2008, 2010, 2011; Wang et al., 2006; Del Galdo et al., 2008; Lee et al., 2014a,b, 2019; Reese et al., 2014a). However, in endothelial cells (that express caveolin-1 at high levels), in some studies CSD appears to act as a surrogate, whereas in others it appears to act as a competitor of caveolin-1 (Chidlow et al., 2009; Tahir et al., 2009). This dual function of CSD has been suggested previously by many authors (Gratton et al., 2003; Chidlow et al., 2009; Tahir et al., 2009).

We recently showed that several readouts associated with AngII treatment and transverse aortic constriction in young mice, including tyrosine kinase activation, microvascular hyperpermeability, organ fibrosis, deficits in ventricular function, and myocyte hypertrophy, can be suppressed by concomitant CSD treatment (Pleasant-Jenkins et al., 2017; Chinnakkannu et al., 2018). However, a treatment that would be useful in human patients should be able to reverse pre-existing disease. Therefore, we have explored the effects of CSD on pre-existing pathologies in several organs in aged mice. Our studies demonstrate that systemic delivery of CSD reverses a variety of aging-associated pathologies. These data are particularly reliable and striking because they demonstrate that the beneficial effects of CSD are therapeutic.

Materials and Methods

Animals

Wild-type C57BL/6J male mice (3 months) were purchased from Jackson Laboratory (Bar Harbor, Maine). For studies with aged mice, an equal number of male and female 18-month-old mice were obtained from local colonies or from the National Institute of Aging Aged Rodent Colony. Mice were maintained at a constant room temperature (20–22°C) with free access to water and food under a 12-hour light/dark cycle. All animal experiments were covered under a protocol approved by the Medical University of South Carolina Institutional Animal Care and Use Committee and were performed twice.

In each figure, data were obtained from three mice per group (containing both male and female mice). These results were confirmed in two additional experiments performed with two or three mice per group. Overall, studies were performed with seven or eight mice per condition, including both male and female mice.

CSD Peptide Treatment

CSD (amino acids 82–101 of caveolin-1, DGIWKASFTTFTVTKYWFYR-NH2) was purchased from Elim Biopharmaceuticals (Hayward, CA). In total, 100 μl of CSD (80 µmol/kg in DMSO diluted 100-fold to a final 0.8 µmol/kg of 1% DMSO in water) or 1% DMSO vehicle was injected daily intraperitoneally for 4 to 6 weeks. This approach to CSD treatment in vivo is the same as we have used in previous studies (Pleasant-Jenkins et al., 2017; Chinnakkannu et al., 2018) and very similar to what other groups have done (Gratton et al., 2003; Bernatchez et al., 2005; Marudamuthu et al., 2019).

Heart function was evaluated by echocardiography, and the mice were sacrificed under deep anesthesia. Mice were systemically perfused via the left ventricle (LV) with PBS, and heart, kidney, and brain tissues were processed for Western blot and histochemical studies.

Echocardiography

Echocardiography (Echo) was performed for young (3-month-old) and aged (18-month-old) mice using a Vevo2100 imaging system (VisualSonics, Toronto, Canada) as previously described (Balasubramanian et al., 2015; Pleasant-Jenkins et al., 2017). Both CSD- and vehicle-treated aged mice were evaluated. Briefly, isoflurane anesthesia was used, and heart rate was maintained at 400–500 beats per minute. Two-dimensional B- and M-mode analyses were performed using the 22- to 55-MHz linear transducer probe. M-mode images of the parasternal short-axis (PSAX) view at papillary level were used to calculate ejection fraction (EF) and fractional shortening (FS) using VisualSonics 1.2.0 software. B-mode images of the parasternal long axis were used to calculate cardiac output (CO) and stroke volume (SV) and end-diastolic volume (EDV). Tissue Doppler was used in the short-axis view at the papillary muscle level to obtain velocity of posterior LV wall motion. In total, 5–10 cycles were recorded to calculate isovolumic relaxation time (IVRT).

Western Blotting

Western blot analyses of LV, kidney, and brain tissue were performed as previously described (Balasubramanian et al., 2015; Pleasant-Jenkins et al., 2017). The radioimmunoprecipitation assay buffer (RIPA)-soluble fractions of each tissue were used for the detection of IgG heavy chain (microvascular leakage) and tyrosine kinase activation. The RIPA-insoluble fractions were used for the detection of Col I (fibrosis). In both fractions, total actin served as the loading control.

Histochemistry

Picrosirius red staining for the detection of pathologic extracellular matrix protein deposition (fibrosis) in the heart and kidney was performed as previously described (Balasubramanian et al., 2012; Pleasant-Jenkins et al., 2017). For quantitation of the data in terms of collagen volume fraction, at least four random fields from each stained tissue sample were viewed under polarized light and the quantified using SigmaScan software. Images were also collected using bright-field microscopy.

Myocyte Hypertrophy

Mouse hearts were fixed with 4% formaldehyde and embedded in paraffin. The 7-μm-thick LV sections were cut, stained with hematoxylin and eosin, imaged using a 20× lens, and photographed. The circumference of myocytes in five random fields was traced and quantified in terms of their cross-sectional area using SigmaScan software.

Statistical Analyses

Values are presented as means ± S.D. Differences were analyzed between groups using one-way ANOVA followed by a post hoc Tukey’s multiple comparison to determine statistical significance. The experiments shown here were performed with three or four mice per group and were repeated two additional times with similar results in male and female mice.

Results

Our previous work using young mice infused with AngII showed microvascular leakage and interstitial fibrosis in the heart and kidney that was suppressed by daily intraperitoneal injections of CSD delivered concomitantly with the AngII (Chinnakkannu et al., 2018). These previous studies were not designed to reveal whether CSD would reverse pre-existing microvascular leakage and/or fibrosis. Here, to demonstrate the outstanding therapeutic potential of CSD, we have used aged mice (18 months old) to demonstrate that CSD treatment reverses a variety of pre-existing pathologies in the heart, kidney, and brain. All data presented contain both sexes.

Reversal by CSD of Pre-existing Microvascular Leakage and Fibrosis in the Aged Heart and Kidney

We first evaluated microvascular leakage [in terms of IgG heavy chain (IgGH)] and fibrosis (in terms of Col I) in heart and kidney tissue from aged mice (Fig. 1A) compared with young mice (3 months old). Both IgGH and Col I levels in the heart and kidney were severalfold higher in aged mice. When the aged mice were treated for 6 weeks with daily intraperitoneal injections of CSD, both IgGH and Col I levels were dramatically reduced almost down to the levels in healthy, young mice. To further validate the reduced fibrosis in CSD-treated aged mice, heart and kidney tissue sections were stained with picrosirius red and the collagen volume fraction determined (Fig. 1B). Similar to Col I levels, picrosirius red staining in aged mice was substantially increased in both the heart and kidney. This increase was reversed by CSD almost down to the level in young mice.

Fig. 1.

Fig. 1

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Microvascular leakage and fibrosis in the aged mouse heart and kidney and reversal by CSD. Young C57/Bl6 mice (3 months) and aged C57/Bl6 mice (18 months) were injected intraperitoneally daily for 6 weeks with saline vehicle or CSD (0.8 µmol/kg in 100 µl). (A) Microvascular leakage was evaluated by Western blot in terms of mouse IgGH and IgGL(using anti-mouse IgG antibody) in the RIPA-soluble fraction from heart and kidney tissue of carefully perfused mice. Fibrosis was evaluated in terms of the level of Col I in the RIPA-insoluble fraction. Actin was the loading control for both fractions. In the quantification graphs (three mice per group), significant changes are shown as ***P < 0.001 and **P < 0.01 between young and aged mice and ^^^P < 0.001 and ^^P < 0.01 between aged mice with and without CSD treatment. (B) Representative examples of picrosirius red staining of heart and kidney tissue sections in the indicated categories are shown and were quantified (three mice per group) as described in the Materials and Methods. Significant changes are shown as ***P < 0.001 and **P < 0.01 between young and aged mice and ^P < 0.05 between aged mice with and without CSD treatment. Values are shown as means ± S.D.

Reversal by CSD of Tyrosine Kinase Activation in the Aged Heart and Kidney

Because tyrosine kinase activation (phosphorylation) has been implicated in vascular hyperpermeability as a result of its effects on junctional proteins, we examined the effect of CSD on the activation of receptor and nonreceptor tyrosine kinases. We observed in both heart and kidney (Fig. 2) a major aging-associated increase in the specific activity of tyrosine kinases. We further observed an increase in the total level of c-Src, but not of PDGFR and Pyk-2. The aging-associated changes in tyrosine kinase level and activation were almost completely reversed by CSD (Fig. 2).

Fig. 2.

Fig. 2

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Tyrosine kinase activation in the aged mouse heart and kidney and reversal by CSD. The same mouse tissue extracts used in Fig. 1 were also used in Fig. 2. The RIPA-soluble fraction was analyzed by Western blot for tyrosine kinase activation using antibodies specific for phosphorylated tyrosine residues in PDGFR (α-Y849/β-Y857), c-Src Y426, and Pyk2 Y402 and antibodies to detect the total levels of these kinases. Actin was the loading control. The quantification graphs (three mice per group) represent the levels of activated kinases normalized to the total level of each kinase. Significant changes are shown as ***P < 0.001 and *P < 0.05 between young and aged mice and ^^^P < 0.001, ^^P < 0.01, and ^P < 0.05 between aged mice with and without CSD treatment. Values are shown as means ± S.D.

CSD Improves Ventricular Structure/Function in Aged Mice

Given the beneficial effects of CSD on microvascular leakage, fibrosis, and tyrosine kinase signaling in the heart, we evaluated the effects of CSD on ventricular function (EF, FS, CO, SV, EDV, and IVRT) by Echo. No significant changes in CO, SV, or EDV were observed associated with either aging or CSD treatment (not shown). However, for FS and SV, although no significant difference was observed between young and aged mice, CSD had a positive effect on these parameters (Fig. 3). Finally, for IVRT (a measurement of diastolic function), a prolonged ventricular relaxation time was associated with aging and was reversed by CSD (Fig. 3).

Fig. 3.

Fig. 3

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CSD improves ventricular function in aged mice. M-mode echo measurements in PSAX view were used to quantify changes in EF and FS. Tissue Doppler measurements in PSAX were used to measure IVRT. Statistically significant changes are shown as ***P < 0.001 between young and aged mice and ^P < 0.05 between aged mice with and without CSD treatment (seven young mice, seven aged mice, three aged mice treated with CSD). Values are shown as means ± S.D. For the statistically significant changes, powers are as follows: IVRT Young vs. Aged, 1.00; IVRT Aged vs. Aged + CSD, 0.81; EF Aged vs. Aged + CSD, 0.91; and FS Aged vs. Aged + CSD, 0.91.

Because young mice treated with AngII undergo cardiac hypertrophy that is reversed by CSD (Chinnakkannu et al., 2018), we evaluated both cardiomyocyte cross-sectional area and the heart weight/body weight (HW/BW) ratio in aged mice. Cardiomyocyte cross-sectional area was increased in aged mice, and this increase was suppressed by CSD (Fig. 4). HW/BW ratio was not significantly increased in aged mice compared with young mice; nevertheless, the beneficial effect of CSD on the HW/BW ratio in aged mice approached significance.

Fig. 4.

Fig. 4

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CSD reverses cardiac hypertrophy associated with aging. LV tissue sections from the indicated mice were stained with hematoxylin and eosin. Cardiomyocyte cross-sectional area was quantified by measuring at least 40 cardiomyocytes per mouse, three mice per group, using SigmaScan Pro image analysis. Cardiac hypertrophy was also evaluated in terms of the HW/BW ratio. Statistical significance is shown as ***P < 0.001 between young and aged mice and ^P < 0.05 between aged mice with and without CSD treatment (four young mice, five aged mice, five aged mice treated with CSD). Values are shown as means ± S.D. For the statistically significant changes, powers are as follows: CSA Young vs. Aged, 1.00; and CSA Aged vs. Aged + CSD, 0.92. The comparison HW/BW Aged vs. Aged + CSD approached significance with a power of 0.75. CSA, cross-sectional area per cell.

Aging and CSD Have Similar Effects on the Mouse Brain as on the Heart and Kidneys

Because of the importance of microvascular leakage in aging-associated neurodegenerative diseases, we also explored the effects of CSD on the aged brain (Fig. 5). The results obtained were similar to those in the heart and kidney. Compared with young mouse brain, there were profound increases in microvascular leakage and fibrosis in aged mice that were reversed by CSD. There was also a major increase in the activation (i.e., specific activity) of PDGFR and Pyk2. For c-Src, there was also an increase in the level of the activated form of the kinase; however, this is due to a significant increase in total c-Src with no increase in specific activity. Again, CSD treatment reversed these pre-existing alterations in tyrosine kinase activation and total levels almost down to what was observed in healthy, young mice.

Fig. 5.

Fig. 5

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Microvascular leakage, fibrosis, and tyrosine kinase activation in the aged mouse brain are reversed by CSD. Mice were treated as described in Fig. 1, except that CSD injections were for 4 weeks. Brain tissue extracts were prepared, and Western blot analyses were performed for microvascular leakage, fibrosis, and phosphorylated and total tyrosine kinase levels (three mice per group). Quantification graphs for levels of fibrosis and microvascular leakage are presented as in Fig. 1, and kinase activation graphs are normalized to total levels of each kinase as in Fig. 2. Significant changes are shown as ***P < 0.001 and **P < 0.01 between young and aged mice and ^^^P < 0.001, ^^P < 0.01, and ^P < 0.05 between aged mice with and without CSD treatment. Because the specific activity of c-Src does not differ significantly among the groups, we also show a quantification graph for total c-Src levels in which significant changes are shown as **P < 0.01 between young and aged mice and ^P < 0.05 between aged mice with and without CSD treatment. Values are shown as means ± S.D.

In summary, systemic treatment with CSD has a profound effect in reversing aging-associated microvascular leakage, fibrosis, and tyrosine kinase activation in multiple organs.

Discussion

We previously demonstrated the beneficial effects of CSD on fibrosis and microvascular leakage in the heart and kidneys and on heart function in models in which disease was induced in young mice by AngII infusion or transverse aortic constriction (Pleasant-Jenkins et al., 2017; Chinnakkannu et al., 2018). CSD treatment in these models was prophylactic, i.e., the treatment began concomitantly with the induction of disease. Here, we are taking a novel therapeutic approach in which aged mice that already have developed robust microvascular leakage and fibrosis are then treated with CSD, resulting in the reversal of these pre-existing pathologies. Additionally, the current studies now include the brain, and analogous results are obtained for the heart and kidney. Therefore, these studies demonstrate the likely clinical relevance of CSD to a range of aging-associated diseases.

In the current study, we have made several novel observations on the biochemistry, structure, and function of multiple organs (heart, kidney, brain) in aged mice (18 months old). There is a striking uniformity of effects: 1) In all of these organs, microvascular leakage and interstitial fibrosis is greatly increased compared with young mice. 2) These pathologies are almost completely reversed by CSD down to the level in healthy, young mice. 3) In all of these organs, the levels of activated tyrosine kinases (both receptor and nonreceptor) are greatly increased with aging. These increases are almost completely reversed by CSD down to the levels observed in healthy, young mice, which in concert with the literature suggests that the activation of these kinases results in microvascular leakage and fibrosis. 4) In studies of organ function, cardiac hypertrophy in aged mice was accompanied by an increase in IVRT, indicating diastolic dysfunction. CSD improved diastolic function by reversing myocyte hypertrophy and the increase in IVRT. In addition, CSD also enhanced ventricular function by increasing EF and FS despite the fact that aging had not altered these parameters. Throughout these studies, similar results were obtained using both male and female mice.

Figure 6 summarizes our overall interpretation of the results of this study. We propose a series of processes that occur during aging in multiple organs (heart, kidney, brain): tyrosine kinase activation plays a central role in this model. When tyrosine kinases are activated (possibly due to an increased ROS:NO ratio), they phosphorylate endothelial junctional proteins in adherens junctions, tight junctions, and gap junctions, leading to a decrease in junctional integrity and a loss of barrier function, resulting in microvascular leakage. Each step in this process has been shown to occur in aged mice: tyrosine kinase activation (Barrett et al., 1999; Frank and Eguchi, 2003; Frank et al., 2003; Droge, 2005) due to an altered ROS:NO ratio (Frank et al., 2000; Cui et al., 2012; Zhang et al., 2016; Masuda et al., 2019), phosphorylation of junctional proteins (van Buul et al., 2005), and loss of junctional integrity (Frank et al., 2003; van Buul et al., 2005).

Fig. 6.

Fig. 6

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Summary of data and their implications. Both aging and AngII infusion have similar effects on tyrosine kinases, microvascular leakage, inflammation, and fibrosis. We propose that the effect on tyrosine kinases is primary (thick arrow) and is upstream from the effect on microvascular leakage, which in turn is upstream from the effect on inflammation and fibrosis. Our work and the literature have shown that CSD directly inhibits a variety tyrosine kinases, microvascular leakage, inflammation, and fibrosis.

In turn, microvascular leakage (Lakatta and Levy, 2003; Brandes et al., 2005; Oakley and Tharakan, 2014) promotes inflammation and fibrosis by allowing profibrotic factors (growth factors and cytokines) and monocytes to enter the tissue (Reddy et al., 1995; Oakley and Tharakan, 2014; Foulquier et al., 2018). These factors induce Col I overexpression and deposition (fibrosis), which in the heart affects ventricular compliance (Weis, 2008; Boyle et al., 2011; Rosenberg, 2014; Saez and Vilchez, 2014; Piek et al., 2016; Toba and Lindsey, 2019).

The beneficial effects of CSD are likely to involve effects on all highlighted parameters in Fig. 6 (tyrosine kinase activation and levels, microvascular leakage, fibrosis). When aged mice are treated systemically with CSD, the pre-existing microvascular leakage in heart, kidney, and brain is reversed almost down to the level observed in young, healthy mice. Given our results on receptor and nonreceptor tyrosine kinases and the literature, it is likely that the ability of CSD to inhibit specific tyrosine kinases (Li et al., 1996; Bucci et al., 2000; Vargas et al., 2002; Jasmin et al., 2006; Fang et al., 2007; Chinnakkannu et al., 2018) contributes to its beneficial effects on the integrity of the microvasculature (Bucci et al., 2000). Furthermore, effects of CSD on the ROS:NO ratio due to its regulation of endothelial nitric oxide synthase coupling and of NADPH oxidase (Karuppiah et al., 2011; Chen et al., 2014) may also contribute to the regulation of tyrosine kinases by CSD. The fact that the systemic delivery of CSD affects microvascular leakage in multiple organs strongly suggests that CSD is universally regulating barrier function, either through a direct effect on endothelial cells or possibly on perivascular cells such as pericytes, smooth muscle cells, and glial cells (Lendahl et al., 2019; Liu et al., 2020).

We have previously shown both in vivo and in vitro that CSD inhibits inflammation in multiple models and organs. The ability of inflammatory cells (monocytes) isolated from humans or mice with fibrotic disease to migrate in vitro was enhanced as a result of decreased levels of caveolin-1 and was suppressed by CSD (Tourkina et al., 2010, 2011; Lee et al., 2014a, 2015, 2017; Reese et al., 2014b; Chinnakkannu et al., 2018). Likewise, the accumulation of monocytes in affected tissues in vivo was suppressed by CSD. The ability of CSD to reverse aging-associated microvascular leakage supports the idea that CSD regulates inflammation both by its effects on the integrity of the microvasculature and by its effect on the response of monocytes to chemotactic stimuli.

The ability to overexpress Col I by myofibroblasts from humans or mice with fibrotic disease is also due to decreased levels of caveolin-1 in these cells and is suppressed by CSD acting as a surrogate for caveolin-1 (Tourkina et al., 2005, 2008, 2010, 2011). Similarly, we show here that fibrosis is greatly increased in aged heart, kidney, and brain and that this increase is reversed by CSD. This effect, combined with our observations on the effects of CSD on tyrosine kinases (Figs. 2 and 5) (Pleasant-Jenkins et al., 2017; Chinnakkannu et al., 2018), suggests that Col I overproduction in aged tissues is regulated in part through the inhibition of tyrosine kinases by CSD. This viewpoint is supported by our earlier work with the tyrosine kinase inhibitor dasatinib (Balasubramanian et al., 2015) and the work of others on the kinase inhibitor nintedanib (Wollin et al., 2019). It is also likely that the inhibition of microvascular leakage contributes to the inhibition of fibrosis by attenuating the entry of profibrotic serum proteins and of inflammatory cells into damaged tissue.

Our studies of organ function focused on the heart. Echo analyses in control aged mice show a similar EF and FS to young, healthy mice; however, IVRT is increased (Fig. 3). Thus, cardiac function in aged mice is reminiscent of diastolic heart failure in human patients, known as heart failure with preserved ejection fraction, the most common form of heart failure in the elderly. In heart failure with preserved ejection fraction, increased fibrosis/stiffness results in a decreased ability of the ventricle to relax (Weber et al., 1992; Gaasch and Zile, 2004; Gradman and Wilson, 2009; Zile et al., 2015). We find that CSD treatment of aged mice reversed the pathologic change in IVRT, as expected from the fact that it reduced the level of Col I. The decreased fibrosis also likely accounts for the improved EF and FS that we observed in aged mice treated with CSD.

Interestingly, when we examined the aged brain, we observed analogous results to the heart and kidney in that there was robust microvascular leakage, fibrosis, and tyrosine kinase activation that was reversed by CSD. This is a rich area for future study because the recent literature indicates that microvascular leakage is an early and key step in a range of neurodegenerative diseases, including Alzheimer disease (Sweeney et al., 2018). In addition, microvascular leakage may be important in the progression of glioma because these metastatic cells tend to grow along the microvasculature (Dubois et al., 2014).

Comparison of CSD with a blockbuster drug (nintedanib) already being used to treat fibrotic diseases in humans suggests that the drug development of CSD will be feasible. As described above, both CSD and nintedanib inhibit a wide range of kinases. When nintedanib is used in mouse model systems, a dose of at least 111 μmol/kg is required to suppress heart and lung fibrosis (Wollin et al., 2014; Ackermann et al., 2017; Rol et al., 2019). As CSD is effective at 0.8 μmol/kg (and may be effective at lower doses), CSD is beneficial at doses at least 139-fold lower on a molar basis than nintedanib, indicating the great potential for CSD drug development. In summary, our results on the ability of CSD to reverse a variety of aging-associated pathologies in multiple organs suggest that versions of CSD may be developed to treat a variety of diseases that currently have few, if any, treatment options. An additional, favorable feature of CSD is that it is a natural, biologic inhibitor of signaling via specific tyrosine kinases. Although synthetic chemical tyrosine kinase inhibitors such as nintedanib and dasatinib have been suggested as therapeutics in other diseases, these drugs suffer from toxicity issues and side effects (Varga et al., 2015; Medeiros et al., 2018; Kato et al., 2019). In contrast, as a biologic, CSD is unlikely to exhibit significant toxicity or immunogenicity.

Acknowledgments

Authorship Contributions

Participated in research design: Kuppuswamy, Hoffman.

Conducted experiments: Chinnakkannu, Reese.

Performed data analysis: Kuppuswamy, Hoffman.

Wrote or contributed to the writing of the manuscript: Kuppuswamy, Hoffman.

Footnotes

This work was supported by a Sponsored Research Agreement from Lung Therapeutics, Inc., and by a College of Medicine Enhancement of Team Science grant from the Medical University of South Carolina.

No author has an actual or perceived conflict of interest with the contents of this article.

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