Main Research Findings

1) Hexarelin is a functionally potent and chemically stable compound that has the potential to act as a therapeutic agent for various cardiovascular diseases.

2) Treatment with hexarelin was found to reduce the formation of abdominal aortic aneurysm by inhibiting the activation of phenotype switch and inflammasome.

 

Selected Data

1) This review article, published by the research team of Mao et al, synthesizes findings from a broad spectrum of primary research studies, each employing diverse and sophisticated methodologies to unravel hexarelin’s effects on the cardiovascular system. As a review, this paper does not present its own experimental methods but rather compiles and discusses the experimental approaches used by the various studies it references. These methodologies collectively encompass in vitro cellular analyses, ex vivo isolated organ preparations, and extensive in vivo animal and human clinical investigations [1].

Many mechanistic insights into hexarelin’s actions were derived from in vitro studies using isolated cell systems. Common models included neonatal rat cardiomyocytes, H9c2 cardiomyocytes, and endothelial cells, which allowed for the direct assessment of hexarelin’s influence on cellular processes such as apoptosis, viability, and proliferation. These studies often involved inducing cellular damage or stress using agents like angiotensin II or doxorubicin to simulate pathological conditions. Cultured cardiac fibroblasts were also utilized to investigate hexarelin’s impact on key fibrotic processes, including collagen synthesis and cellular proliferation. Furthermore, some research employed ex vivo preparations, such as isolated rat papillary muscle and whole perfused or working hearts. These setups permitted precise measurements of inotropic responses, electrophysiological properties, and the hearts’ resilience to ischemia-reperfusion injury in a highly controlled environment, minimizing systemic influences. Additionally, specific human breast carcinoma cell lines, MCF7, T47D, MDA-MB-231, were used in certain receptor binding studies, leveraging their known expression profiles to differentiate hexarelin’s binding characteristics.

A wide array of in vivo animal models was instrumental in translating cellular and isolated organ findings to systemic physiological contexts. Various rat models were extensively used, tailored to specific disease conditions: spontaneously hypertensive rats served as models for cardiac fibrosis and hypertension, while obese Zucker rats provided a model for metabolic syndrome and its associated cardiovascular complications. To delineate the growth hormone (GH)-independent actions of hexarelin, GH-deficient and hypophysectomized rats were employed. Sprague-Dawley rats were a common choice for studies on atherosclerosis. Models of cardiac pathology, such as myocardial infarction induced by left coronary artery ligation and experimental ischemia-reperfusion injury, were crucial for evaluating hexarelin’s cardioprotective, reparative, and anti-remodeling capabilities. These animal studies allowed for the comprehensive assessment of hexarelin’s effects on overall cardiac function, systemic hemodynamics, and the progression of various cardiovascular diseases, alongside the evaluation of long-term treatment outcomes [1].

Although fewer in number within the scope of this review, human clinical studies provided invaluable translational data. These investigations included healthy human volunteers, where hexarelin’s acute hemodynamic and hormonal effects were assessed. More clinically relevant studies involved patients with existing cardiovascular conditions, such as coronary artery disease undergoing by-pass surgery. In these human cohorts, researchers monitored key parameters including left ventricular ejection fraction (LVEF), cardiac output, blood pressure, and heart rate to understand hexarelin’s direct clinical impact on cardiac performance and recovery.

Hexarelin administration strategies varied from acute, single intravenous injections of 2 µg/kg to chronic treatments involving subcutaneous or oral routes for extended periods, for example, 100 µg/kg per day for two weeks or five weeks. These different regimens allowed for the investigation of both immediate and sustained effects. To dissect the molecular mechanisms, various pharmacological tools were employed. Growth hormone secretagogue receptor (GHSR) antagonists, such as d-Lys-3-GH-releasing peptide-6 and BIM28163, were used to block GHSR1a activity, while protein kinase C inhibitors like chelerythrine helped identify signaling pathways involved in hexarelin’s cardioprotective actions [1].

A broad spectrum of sophisticated techniques was utilized to quantify hexarelin’s effects. Cardiac performance was assessed through advanced imaging modalities such as radionuclide angiocardiography, transoesophageal echocardiography, and transthoracic echocardiography to precisely quantify parameters like LVEF, stroke volume, cardiac output, and cardiac index. Hemodynamic measurements included mean arterial pressure, heart rate, systemic vascular resistance index, and central venous pressure. Pathological evaluations, such as infarct size, were determined using triphenyltetrazolium chloride (TTC) staining. Cardiac fibrosis was quantified through direct measurements of collagen deposition and hydroxyproline content, while atherosclerosis progression was assessed by analyzing plaque formation, neointima development, calcium sedimentation in arterial walls, and foam cell formation. At the molecular and cellular levels, studies measured gene and protein expression of various factors. Cellular function was probed by analyzing intracellular Ca2+ transients, L-type Ca2+ currents, and electrophysiological properties like action potential duration. Receptor binding studies utilized radioreceptor assays with labeled hexarelin or fluorescein-conjugated ghrelin. Serum analyses included lipid profiles and nitric oxide levels. Collectively, these diverse and advanced methodologies provided a robust foundation for the comprehensive understanding of hexarelin’s profound cardiovascular actions summarized in the review [1].

2) The study performed by the research team of Jiang et al employed a comprehensive experimental design utilizing an in vivo mouse model of abdominal aortic aneurysm (AAA) to investigate the therapeutic effects and underlying mechanisms of hexarelin. This multi-faceted approach combined animal experimentation with various molecular, histological, and imaging techniques to assess the impact of hexarelin on AAA development, smooth muscle cell (SMC) phenotype, inflammation, and cellular signaling pathways [2].

The primary in vivo model involved 16-week-old male C57BL/6J mice, selected for their suitability in elastase-induced AAA experiments. The AAA model was established via intraluminal elastase perfusion: mice were anesthetized, infrarenal abdominal aortas were isolated, and infrarenal branches were ligated. A 30-gauge needle was used to puncture the aorta near the bifurcation, through which a PE-10 catheter was implanted. Porcine pancreatic elastase at a dose of 1 U/ml in saline was then perfused for 5 minutes at a controlled pressure of 100 mmHg. Following perfusion, the aortotomy was repaired, and the wound was closed. Mice were allowed to recover post-surgery. Hexarelin treatment commenced immediately after elastase infusion, with mice receiving intraperitoneal injections of 200 µg/kg hexarelin dissolved in saline at 50 µg/ml, twice daily for 14 consecutive days. Control mice received an equal volume of saline as a vehicle [2].

To evaluate the extent of AAA formation, ultrasound imaging was performed on day 14 post-surgery after anesthetizing the animals. Researchers measured the proximal non-expanding area of the abdominal aorta and the maximum inner luminal diameter of the infrarenal abdominal aortas. This allowed for quantitative assessment of aortic dilation. Gross in situ pictures of the abdominal aortas were also taken to visually document the aneurysm. Histological examination was critical for assessing tissue integrity. Abdominal aortas were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 5 µm thickness. Slides were dewaxed and boiled in antigen retrieval solution. Elastin degradation, a hallmark of AAA, was specifically evaluated using Verhoeff-Van Gieson staining. The degree of elastin degradation was semi-quantitatively graded from 1, indicating <25% degradation, to 4, indicating >75% degradation, across 6 serial sections per mouse.

The study investigated the impact of hexarelin on SMC phenotype and cell death. Immunofluorescence staining was employed on aortic sections to visualize key cellular markers. Primary antibodies included anti-α-SMA, a contractile SMC marker, which was co-stained with DAPI for nuclear visualization. This allowed for the assessment of SMC contractile phenotype. To identify cell death, the terminal deoxynucleotidyl transferase dUTP nick and labelling (TUNEL) assay was performed on aortic sections, co-stained with anti-α-SMA and DAPI, to quantify the percentage of TUNEL-positive SMCs [2].

Inflammation, a crucial component of AAA pathology, was thoroughly investigated. Immunofluorescence staining was used to quantify the infiltration of inflammatory cells by detecting CD45, a pan-leukocyte marker, and to visualize key inflammatory proteins. These included IL-18 and NLRP3, both central to inflammasome activation, and NF-κB and phosphorylated NF-κB, key components of inflammatory signaling. Western blotting was utilized for quantitative protein analysis of various markers from abdominal aorta lysates. Samples were prepared using a RIPA buffer with protease and phosphatase inhibitors, separated by SDS-PAGE, and transferred to PVDF membranes. Primary antibodies targeted NF-κB, p-NF-κB, MMP-2, NLRP3, caspase-1 (total and cleaved forms), IL-1β, and IL-18. β-actin served as a loading control [2].

 

Discussion

1) The review article completed by Mai et al compiles and presents findings demonstrating hexarelin’s extensive and beneficial cardiovascular actions across various physiological and pathological contexts. The summarized results highlight hexarelin’s direct cardiac effects, its protective roles against various injuries and diseases, and the intricate receptor mechanisms mediating these actions, often showcasing its superior potency compared to ghrelin. Hexarelin consistently exhibits a potent positive inotropic effect, enhancing cardiac contractility. Acute intravenous administration in both human volunteers and animal models leads to a rapid and significant increase in left ventricular ejection fraction (LVEF), cardiac output, and cardiac index. Intriguingly, these improvements in cardiac performance can occur without substantial changes in mean blood pressure or heart rate in some settings. Hemodynamically, hexarelin can reduce wedge pressure while increasing mean arterial pressure and transiently decreasing central venous pressure [1].

At the cellular level, its inotropic effects in ventricular myocytes are time- and concentration-dependent, involving increases in intracellular Ca2+ transients and L-type Ca2+ current, mediated through protein kinase C signaling pathways. Beyond contractility, hexarelin exerts significant anti-apoptotic effects. It has been shown to decrease angiotensin II-induced apoptosis and DNA fragmentation in neonatal rat cardiomyocytes, simultaneously increasing their viability. Furthermore, hexarelin protects H9c2 cardiomyocytes and endothelial cells from doxorubicin-induced apoptosis, thereby promoting cell survival. In models of chronic congestive heart failure, long-term hexarelin treatment successfully alleviates left ventricular dysfunction, pathological remodeling, and cardiac cachexia by suppressing detrimental stress-induced neurohormonal activation and cardiomyocyte apoptosis.

A pivotal finding across multiple studies is hexarelin’s robust cardioprotective capacity against ischemia-reperfusion (I/R) injury. In isolated rat hearts subjected to I/R, hexarelin significantly reduces infarct size, a benefit partially reversible by protein kinase C inhibitors, suggesting a mechanistic link. It also preserves the electrophysiological properties of cardiomyocytes and inhibits apoptosis while promoting cell survival through the modulation of mitogen-activated protein kinase pathways. In in vivo models of I/R, such as those involving Zucker rats, hexarelin effectively counteracts ischemic heart damage, leading to a greater recovery of left ventricular pressure and minimal increases in coronary resistance. Crucially, these protective effects are often GH-independent, as demonstrated by its efficacy in GH-deficient or hypophysectomized animals, and its distinct actions compared to recombinant human GH or GH-releasing hormone [1].

Hexarelin demonstrates significant anti-remodeling and anti-fibrotic properties. In spontaneously hypertensive rats, chronic hexarelin treatment substantially reduces cardiac fibrosis, evidenced by decreased interstitial and perivascular myocardial collagen deposition and lower hydroxyproline content. This is accompanied by a reduction in collagen I and III mRNA and protein expression and a beneficial shift in matrix metalloproteinase (MMP) activity. These actions contribute to attenuating left ventricular hypertrophy, diastolic dysfunction, and high blood pressure. Hexarelin also directly inhibits angiotensin II-induced proliferation and collagen synthesis in cultured cardiac fibroblasts, and reduces TGF-β-induced DNA synthesis and release, thereby disrupting key drivers of fibrotic remodeling [1].

Furthermore, hexarelin exhibits potent anti-atherosclerotic activity. In Sprague-Dawley rats, it suppresses the formation of atherosclerotic plaques and neointima, partially reverses unfavorable serum high-density lipoprotein/low-density lipoprotein cholesterol ratios, and enhances endothelial function by increasing nitric oxide levels and endothelial nitric oxide synthase (eNOS) mRNA expression. Hexarelin also reduces proliferation in vascular smooth muscle cells, decreases calcium sedimentation in the aortic wall, and inhibits foam cell formation induced by oxidized low-density lipoprotein, underscoring its broad protective role against vascular disease. In obese rat models, chronic hexarelin treatment significantly lowers plasma cholesterol levels without altering triglycerides.

The review elucidates that hexarelin’s cardiovascular actions are mediated through a complex interplay of receptors. While it binds to the GHSR1a, a significant portion of its effects are GH-independent. Specific binding sites for hexarelin are found throughout the human cardiovascular system, with the highest concentrations in the ventricles. Two primary cardiac receptor mechanisms are identified. This first of which, cardiac GHSR1a is a receptor expressed in the heart and its activity is modulated by hexarelin, mediating some effects on action potential duration, positive inotropy, and electrophysiological properties. These effects can be abolished by specific GHSR antagonists, confirming GHSR1a involvement. Second, cardiac CD36, which has been identified as a distinct, hexarelin-specific cardiac receptor. Expressed in cardiomyocytes and microvascular endothelial cells, CD36 mediates hexarelin’s actions, such as increased coronary perfusion pressure, which are notably absent in CD36-null mice. Hexarelin’s ability to inhibit proliferation in human breast carcinoma cell lines that lack GHSR1a mRNA further supports CD36 as a key mediator [1].

Comparative studies consistently highlight hexarelin’s superior potency over its natural analogue, ghrelin, in eliciting beneficial cardiovascular effects. In hypophysectomized rats and ghrelin-null mice following myocardial infarction, hexarelin proves more effective in preventing adverse left ventricular changes and improving heart function. While both peptides can induce similar cardioprotective effects against I/R injury, hexarelin often achieves these at significantly lower molar concentrations. This enhanced potency of hexarelin is largely attributed to its stronger interactions with the CD36 receptor, and in part, with GHSRs, making it a highly promising therapeutic candidate for a wide range of cardiovascular pathologies [1].

2) The study’s results comprehensively demonstrate that hexarelin treatment significantly attenuates elastase-induced AAA formation in mice, primarily by preserving SMC contractile phenotype and suppressing inflammatory responses mediated by the NF-κB pathway and inflammasome activation. These findings provide strong evidence for hexarelin’s therapeutic potential in AAA [2].

The first major finding confirmed that hexarelin treatment effectively reduced AAA formation in the elastase-induced mouse model. Echocardiography and in situ imaging at day 14 revealed a significant increase in infrarenal aortic diameter in vehicle-treated mice. In contrast, hexarelin-treated mice exhibited a markedly reduced increase in aortic diameter, with the maximal infrarenal aorta diameter being only slightly elevated compared to baseline, in stark contrast to the substantial dilation seen in the vehicle group. Quantitatively, the percent increase in aortic diameter was significantly lower in hexarelin-treated animals. Histological analysis, particularly Verhoeff-Van Gieson staining, further supported these observations by showing a pronounced decrease in elastin fragmentation and a much lower elastin degradation grade in the aortas of hexarelin-treated mice compared to vehicle controls. These results collectively indicated that hexarelin treatment effectively prevented the progression of experimental AAA [2].

The study then investigated hexarelin’s impact on SMCs, which are critical for aortic wall integrity. It was found that hexarelin treatment preserved the contractile phenotype of SMCs in the aneurysmal aorta. Immunofluorescence staining for α-SMA, a key marker of contractile SMCs, revealed markedly higher α-SMA intensity in the aortas of hexarelin-treated mice compared to the vehicle group. This suggested that hexarelin maintained the contractile function of SMCs, which is often lost during AAA progression. Furthermore, the study elucidated hexarelin’s effect on matrix metalloproteinase-2 (MMP2), a crucial enzyme involved in extracellular matrix degradation and AAA pathology. Immunofluorescence and Western blotting data showed that MMP2 expression was significantly elevated in vehicle-treated aneurysms but substantially reduced by hexarelin treatment. This reduction in MMP2 contributes to the preservation of elastin and the overall structural integrity of the aortic wall. Additionally, TUNEL staining combined with α-SMA labeling demonstrated a significant decrease in the percentage of apoptotic α-SMA-positive SMCs in the hexarelin-treated group, indicating that hexarelin prevented SMC death, a common event in AAA pathogenesis.

A critical aspect of AAA development is robust inflammation. Hexarelin treatment significantly ameliorated the inflammatory response in the aneurysmal aorta. Immunofluorescence staining showed a prominent infiltration of CD45-positive inflammatory cells in the media and adventitia of vehicle-treated mice, which was markedly suppressed in hexarelin-treated animals. The study further delved into the inflammasome pathway, a key driver of inflammation. Hexarelin treatment significantly reduced the expression of IL-18, a cytokine triggered by NLRP3 inflammasome, as confirmed by immunofluorescence. More importantly, the accumulation of NLRP3 inflammasome itself was diminished in both the media and adventitia of hexarelin-treated mice. Western blot analysis corroborated these findings, showing decreased protein levels of NLRP3, as well as the active protease caspase-1 (cleaved caspase-1), which is essential for NLRP3 inflammasome activation. Furthermore, hexarelin treatment led to decreased levels of both pro- and mature IL-1β, another pivotal cytokine released by the NLRP3 inflammasome. These results highlight hexarelin’s capacity to inhibit the NLRP3 inflammasome and its downstream inflammatory mediators, thereby dampening the overall inflammatory cascade in AAA [2].

The NF-κB signaling pathway is a central regulator of inflammation and inflammasome activation. The study demonstrated that hexarelin suppressed the activation of NF-κB signaling. Immunofluorescence staining revealed a slight decrease in total NF-κB expression and a significant inhibition of phosphorylated NF-κB (p-NF-κB) levels in the aortas of hexarelin-treated mice compared to vehicle controls. Western blot analysis confirmed these observations, showing reduced protein levels of both total NF-κB and p-NF-κB in the hexarelin-treated group. This suppression of NF-κB signaling is crucial, as it is a key initiator of inflammatory responses and directly contributes to inflammasome activation and SMC pyroptosis. By inhibiting this pathway, hexarelin likely prevents a cascade of events that exacerbate AAA progression.

In summary, the results collectively illustrate that hexarelin attenuates elastase-induced AAA formation by multiple mechanisms: it preserves the contractile phenotype of SMCs by reducing MMP2 expression and preventing their apoptosis, and critically, it significantly dampens the inflammatory response by suppressing NF-κB signaling and inhibiting NLRP3 inflammasome activation and the subsequent release of pro-inflammatory cytokines like IL-1β and IL-18. These findings highlight hexarelin’s potential as a promising therapeutic agent for the treatment of AAA [2].

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Mao Y, Tokudome T, Kishimoto I. The cardiovascular action of hexarelin. J Geriatr Cardiol. 2014;11(3):253-258. doi:10.11909/j.issn.1671-5411.2014.03.007

[2] Jiang B, Wang M, Li X, et al. Hexarelin attenuates abdominal aortic aneurysm formation by inhibiting SMC phenotype switch and inflammasome activation. Microvasc Res. 2022;140:104280. doi:10.1016/j.mvr.2021.104280

Hexarelin and Cardiac Function

Hexarelin is a synthetically developed growth hormone receptor (GHR) that is known for its ability to efficiently activate the growth hormone secretagogue receptor (GHSR) in a fashion similar to that of the naturally occurring peptide, ghrelin. However, when comparing hexarelin to ghrelin, hexarelin is the more stable and potent compound of the two.

Previous studies show that ghrelin can be administered to improve cardiovascular functioning in terms of cardiac output, ejection fraction, and exercise capacity in rats experiencing chronic heart failure. While ghrelin is successful in treating heart failure it is considered a highly unstable compound, hexarelin on the other hand has shown promise in treating cardiovascular disease on the same level as ghrelin, however, hexarelin is far more stable and much more effective.

Hexarelin and Improved Cardiac Health

In a study conducted by Mao et. Al, hexarelin was administered to various animal subjects in order to observe its effects on apoptosis, ischemia-reperfusion injury, myocardial infarction, cardiac fibrosis, and atherosclerosis.

Neonatal rats were treated with varying doses of hexarelin to examine how the peptide is capable of inhibiting apoptosis in cardiomyocytes. The results of this portion of the study found that hexarelin treatment decreased levels of apoptosis induced by angiotensin II as well as doxorubicin-induced apoptosis. Additionally, it helped to increase the viability of the myocytes and promoted the survival of cardiomyocytes and cardiac epithelial cells.

Zucker rats were treated with 1 micromol/L of hexarelin and then subjected to ischemia for 30 minutes and reperfused for 120 minutes. It was found that with this dose of hexarelin for 30 days the heart damage caused by the ischemia was significantly decreased. The hexarelin treatment promoted cell survival, decreased apoptosis of the cardiomyocytes, and preserved the electrical properties of the myocytes. Furthermore, results showed that in rats treated with hexarelin there was improvement in left ventricle pressure as well as more protection against ischemia and angiotensin II secretion.

Male rats underwent ligation of the coronary artery. Four weeks after the procedure the rats were treated with 100 micrograms/kg/day of hexarelin for two weeks. The results of this portion of the study were very straightforward, it was found that this method of treatment led to a decrease in peripheral resistance, and increases in stroke volume, cardiac output, and cardiac index.

Cardiac fibrosis occurs when there is too much collagen, types I and II, and proteins being deposited throughout the cardiac muscle. Treatment with hexarelin in hypertensive rats over 5 weeks led to a decrease in cardiac fibrosis via a decrease in collagen deposition, and hydroxyproline content in the myocytes. Furthermore, hexarelin treatment decreased blood pressure, left ventricular hypertrophy, and diastolic dystrophy. Hexarelin also decreased the upregulation of TGF-beta expression and secretion caused by angiotensin II.

Atherosclerosis is caused by increased levels of triglycerides and cholesterol. Sprauge-Dawley rats experiencing atherosclerosis were treated with hexarelin. Results found that the formation of atherosclerotic plaques was prohibited. Additionally, hexarelin treatment was seen to lower the ratio of HDLP cholesterol to LDL cholesterol and increase levels of nitric oxide and the mRNA expression of endothelial nitric oxide synthase. While there was little to no change in the triglyceride levels, plasma cholesterol was reduced. This led researchers to conclude that hexarelin is capable of treating atherosclerosis through the regulation of various cholesterol levels (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4178518/).

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