Regenerative Medicine for Critical Limb Ischemia

1. Regenerative Medicine for Critical Limb Ischemia (CLI) Treatment Ischemia is a medical term describing restricted blood flow, which compromises the supply of oxygen and nutrients to an organ. Critical Limb Ischemia (CLI) is defined by reduced blood flow to lower extremities - particularly legs, leading to pain, ulcers, and loss of limbs. It is a severe form of peripheral artery disease (PAD). The disorder occurs due to the narrowing or blockage of arteries in the lower limbs. The updated guidelines now refer to critical limb ischemia as “chronic limb-threatening ischemia”. This condition is chronic in nature and develops over months to years. What is a no-option CLI? Revascularization is a surgical procedure that is recommended to prevent loss of limb. However, not all patients are suitable candidates for this procedure, and in some cases, it proves ineffective. When conventional treatments and revascularization fail, the condition is referred to as no-option CLI. Patients in this category face a high risk of limb amputation and require alternative therapeutic strategies. (Note: Stem cell treatment has shown positive results in no-option CLI) What are the outcomes of CLI? Approximately 10-40% patients with critical limb ischemia are at a high risk of limb amputation within a period of one to five years of developing the disorder. A study demonstrated that the risk of cardiovascular-associated mortality is 5.9 times higher in patients with critical limb ischemia. These statistics highlight the urgency of timely and effective treatment to improve patient outcomes. Are acute limb ischemia and chronic limb ischemia the same? No. Acute limb ischemia develops within two weeks, whereas chronic critical limb ischemia develops slowly over time, requiring months to progress to the reduced blood flow. The therapeutic interventions of critical limb ischemia emphasize managing the causative factors, relieving pain, and slowing disease progression. Minimally invasive and surgical techniques reinstate blood flow but are not suitable for everyone. The high mortality rate of critical limb ischemia has urged scientists to develop an advanced treatment that can address the underlying pathology. Stem Cell Therapy has been the focus of the scientific community for years. It is hailed as the next- generation therapy that is redefining the treatment approach with its regenerative potential. Recent years have witnessed the growth of stem cell therapy for diverse conditions, promoting its application for critical limb ischemia. The evolving technology has facilitated personalized Stem Cell Therapy for critical limb ischemia. It designs customized treatment plans accounting for the patient’s attributes and disease characteristics. This paves the way for precision medicine, transforming the lives of critical limb ischemia patients. Diagnosis Early and accurate diagnosis is crucial to prevent the progression of disease to an advanced stage. Clinicians use a combination of physical evaluation and imaging tests. Physical Examination: Clinician first examines legs and feet to detect the symptoms of critical limb ischemia. Skin temperature, sensitivity to touch, skin color, and pulse in limbs can indicate critical limb ischemia. Ankle-Brachial Index (ABI): Critical limb ischemia is diagnosed by this non-invasive test. The test measures the blood pressure in the legs and compares it to that in the arms to calculate an ankle (legs)- www.advancells.com +91- 9654321400

2. brachial (arm) index (ABI). ABI ranges between 0.9 and 1.4. An index of 0.9 implies low blood pressure in the legs. This technique also measures ankle pressure (AP). AP less than 50 mm Hg indicates critical limb ischemia. Toe-Brachial Index (TBI): It is similar to ABI and calculates the blood pressure ratio in the toe to that in the arm. It is beneficial in diabetic patients who show calcium deposition in arteries which can make the ABI unreliable. Transcutaneous Oxygen Pressure (TcPO2): This non-invasive technique determines the oxygen supply in the arteries, indicating the severity of ischemia and the capacity of wound healing. Small sensors or electrodes are placed on the skin which measure the oxygen tension. Duplex Ultrasound: It is a non-invasive real-time imaging technique that assesses the blood flow. The instrument emits sound waves and captures them after they bounce off the blood vessels. The obstruction or narrowing of vessels changes the blood flow, which is reflected in the modified frequency of the received sound waves. Angiography: Angiography involves injecting a contrast dye into the blood vessels and visualizing them through imaging techniques. Computer Tomography Angiography (CTA) and Magnetic Resonance Angiography (MRA) are frequently used imaging techniques. They show the flow of the contrast dye through vessels and thus enable identification of vessel narrowing or obstruction. Early Signs & Symptoms of CLI Disease Clinicians generally use a combination of three critical limb ischemia symptoms for diagnosis. Rest Pain: It is the most prominent sign of critical limb ischemia, characterized by pain in legs and feet while a person is not moving. Non-Healing Wounds: Ulcers or sores on the legs or feet that don’t heal. Gangrene: Tissue death due to lack of oxygen, often requiring amputation. Rest pain especially increases at night when the patient is in a supine position and the contribution of gravity to leg arterial pressure is low. Additionally, the critical limb ischemia symptoms have been termed as 5Ps to facilitate diagnosis.  Pain: Pain or cramping at rest  Pallor of skin: Shiny, hairless, or discolored skin, along with toenail thickening  Pulselessness: Diminished pulses in legs and feet due to reduced blood flow  Paresthesia: Tingling or numbness in the affected leg or foot  Pokilothermia: Coldness in the Limb Available Types of Critical Limb Ischemia Many different classification systems have emerged since the 1950s to classify the various stages of critical limb ischemia. Appropriate classification is a key determinant in strategizing the suitable treatment plan and delaying the disease progression. The most recent classification system, WIFi, was established by the Society of Vascular Surgery in 2014. It categorizes the disorder according to:  Wound (W) www.advancells.com +91- 9654321400

3.  Ischemia (I): Measured by ankle-brachial index (ABI), ankle pressure (AP), and transcutaneous oxygen pressure (TcPO2)  Foot infection (Fi) It defines the following classification of CLI into grades: Grade 0  Wound- no wound  Ischemia-ABI ≥ 0.80, AP > 100, TcPO2≥ 60  Foot infection- no sign of infection Grade 1  Wound- minor tissue loss  Ischemia- ABI: 0.60-0.79, AP: 70-100, TcPO2: 40-59 Foot infection-local infection ≤ 2 cm2 Grade 2   Wound- major tissue loss  Ischemia- ABI:0.40-0.59, AP: 50-70, TcPO2 30-49 Foot infection-local infection ≥ 2 cm2 or in deep tissue Grade 3   Wound- extensive tissue loss  Ischemia-ABI ≤ 0.39, AP < 50, TcPO2 < 30  Foot infection- systemic infection Moreover, ICD 10 Code for critical limb ischemia have mentioned different diagnostic codes for the disorder:  I70.22: Atherosclerosis or critical limb ischemia in arteries with rest pain  I70.23: Atherosclerosis or critical limb ischemia in arteries of right leg with ulceration  I70.24: Atherosclerosis or critical limb ischemia in arteries of left leg with ulceration  I70.26: Atherosclerosis or critical limb ischemia in arteries with gangrene What Causes CLI? Critical limb ischemia is primarily caused by plaque build-up inside the arteries, also called atherosclerosis. It triggers angiogenesis pathways that increase the artery diameter to restore optimal blood supply. With time, artery dysfunctions and triggers inflammation, increasing vessel permeability, and stimulating free radical production. These pathological changes lead to fluid retention and tissue swelling, which further elevate pressure on already narrowed arteries, worsening blood flow and accelerating ischemic damage. Common critical limb ischemia causes are:  Age  Diabetes www.advancells.com +91- 9654321400

4.  High Blood Pressure (Hypertension)  High Cholesterol (Hyperlipidemia)  Obesity  Smoking  Family History of CLI  Sedentary Lifestyle What are available treatments for CLI ? Available Critical limb Ischemia Treatments aim to relieve pain, heal ulcers, and prevent disease progression. Surgical interventions are not suitable for all patients and show limited effectiveness. However, the risk of amputation rate remains considerable with these therapies. In an attempt to explore novel and effective alternatives, scientists have ventured into the field of regenerative medicine. Stem cells, the backbone of regenerative medicine, have various beneficial therapeutic properties in treating critical limb ischemia. The following pathways detail the mechanisms through which stem cell treatment for critical limb ischemia targets the underlying pathways and induces regeneration:  Stem cells transform into endothelial cells of blood vessels to form more arteries.  The cross-talk between stem cells and endothelial cells stimulates the migration and growth of endothelial cells to stimulate artery formation and reduce the permeability of blood vessels.  These cells also increase the expression of proteases such as MMP9, MMP2, MT1-MMP, etc., and release growth factors like VEGF, FGF2, and IL8 that drive the formation of vessels and restore blood flow.  The treatment also suppresses inflammation by secreting PGE2, sTNFR, TSG6, IL8, CXCL5, and CXCL6.  Stem cells also stimulate the induction of M2 macrophages and increase the number of regulatory T cells to modulate the immune system in favor of reparative pathways. Frequently Asked Questions Q1. How is stem cell therapy for critical limb ischemia better than the present treatment modalities? Conventional treatments employ revascularization to prevent loss of limb. It involves either a minimally invasive endovascular technique or a surgical bypass. The procedure uses a healthy vessel to reroute blood around a blocked artery. Stem cell treatment for critical limb ischemia creates new vessels without requiring surgical procedures to restore the blood flow to limbs. Q2. Is stem cell therapy effective in no-option CLI? Yes. Multiple clinical studies have evaluated stem cell therapy in no-option CLI patients and reported positive outcomes. One notable clinical trial showed that mesenchymal stem cell (MSC) administration led to an amputation-free survival rate of 92.58% with significant improvements in wound healing and blood flow. Q3. Which stem cells are used for CLI treatment? Clinical trials, either completed or ongoing, primarily employ MSCs to treat critical limb ischemia. Both allogeneic and autologous MSCs from different sources, including bone marrow, umbilical cord, and adipose tissue, have shown substantial improvements in critical limb ischemia. www.advancells.com +91- 9654321400

5. Q4. Is stem cell treatment for critical limb ischemia safe? Over 25 clinical trials have assessed the safety and efficacy of Stem Cell Therapy—particularly MSCs— for critical limb ischemia. They have demonstrated that complications in the stem cell treatment group and the group treated with conventional therapies remained the same. None of them has reported any adverse events occurring due to the stem cell therapy for critical limb ischemia. www.advancells.com +91- 9654321400